Isotopologues of 5-azacytidine

ABSTRACT

The present disclosure provides pharmaceutical compositions comprising cytidine analogs for oral administration, wherein the compositions release the cytidine analog substantially in the stomach. Also provided are methods of treating diseases and disorders using the oral formulations provided herein.

I. CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to, and is a continuation of U.S.patent application Ser. No. 16/582,779, filed Sep. 25, 2019, which is acontinuation of U.S. patent application Ser. No. 16/244,014, filed Jan.9, 2019, which is a continuation of U.S. patent application Ser. No.15/835,088, filed Dec. 7, 2017, which is a divisional of U.S. patentapplication Ser. No. 14/788,606, filed Jun. 30, 2015, entitled “OralFormulations of Cytidine Analogs and Methods of Use Thereof”, which is acontinuation of U.S. patent application Ser. No. 14/463,424, filed Aug.19, 2014, entitled “Isotopologues of 5-Azacytidine”, which is acontinuation of U.S. patent application Ser. No. 12/466,213, filed May14, 2009, entitled “Oral Formulations of Cytidine Analogs and Methods ofUse Thereof”, which claims priority to U.S. Provisional PatentApplication Nos. 61/053,609, filed May 15, 2008; 61/201,145, filed Dec.5, 2008; and 61/157,875, filed Mar. 5, 2009, the contents of each ofwhich are incorporated by reference herein in their entireties.

II. FIELD

Provided herein are pharmaceutical formulations comprising cytidineanalogs, or their salts, solvates, hydrates, precursors, and/orderivatives thereof, for oral administration in subjects. Also providedare methods for making the formulations and methods for using theformulations to treat diseases and disorders including cancer, disordersrelated to abnormal cell proliferation, hematologic disorders, andimmune disorders, among others.

III. BACKGROUND

Cancer is a major worldwide public health problem; in the United Statesalone, approximately 570,000 cancer-related deaths were expected in2005. See, e.g., Jemal et al., CA Cancer J. Clin. 55(1):10-30 (2005).Many types of cancer have been described in the medical literature.Examples include cancer of the blood, bone, lung (e.g., non-small-celllung cancer and small-cell lung cancer), colon, breast, prostate, ovary,brain, and intestine. The incidence of cancer continues to climb as thegeneral population ages and as new forms of cancer develop. A continuingneed exists for effective therapies to treat subjects with cancer.

Myelodysplastic syndromes (MDS) refers to a diverse group ofhematopoietic stem cell disorders. MDS affects approximately40,000-50,000 people in the U.S. and 75,000-85,000 subjects in Europe.MDS may be characterized by a cellular marrow with impaired morphologyand maturation (dysmyelopoiesis), peripheral blood cytopenias, and avariable risk of progression to acute leukemia, resulting fromineffective blood cell production. See, e.g., The Merck Manual 953 (17thed. 1999); List et al., J. Clin. Oncol. 8:1424 (1990).

MDS are grouped together because of the presence of dysplastic changesin one or more of the hematopoietic lineages including dysplasticchanges in the myeloid, erythroid, and megakaryocytic series. Thesechanges result in cytopenias in one or more of the three lineages.Patients afflicted with MDS may develop complications related to anemia,neutropenia (infections), and/or thrombocytopenia (bleeding). From about10% to about 70% of patients with MDS may develop acute leukemia. In theearly stages of MDS, the main cause of cytopenias is increasedprogrammed cell death (apoptosis). As the disease progresses andconverts into leukemia, a proliferation of leukemic cells overwhelms thehealthy marrow. The disease course differs, with some cases behaving asan indolent disease and others behaving aggressively with a very shortclinical course that converts into an acute form of leukemia. Themajority of people with higher risk MDS eventually experience bonemarrow failure. Up to 50% of MDS patients succumb to complications, suchas infection or bleeding, before progressing to AML.

Primary and secondary MDS are defined by taking into account patients'prior history: previous treatments with chemotherapy, radiotherapy orprofessional exposure to toxic substances are factors delineatingsecondary MDS (sMDS) from primary MDS. Cytogenetically, one differencebetween the two groups is the complexity of abnormal karyotypes; singlechromosome aberrations are typical for primary MDS, while multiplechanges are more frequently seen in secondary disorders. Some drugs mayhave specific targets such as hydroxurea for 17p and topoisomerasesinhibitors for 11q23 and 21q22. The genetic changes in the malignantcells of MDS result mainly in the loss of genetic material, includingprobable tumor suppressor genes.

An international group of hematologists, the French-American-British(FAB) Cooperative Group, classified MDS into five subgroups,differentiating them from acute myeloid leukemia. See, e.g., The MerckManual 954 (17th ed. 1999); Bennett J. M., et al., Ann. Intern. Med.,103(4): 620-5 (1985); and Besa E. C., Med. Clin. North Am. 76(3):599-617 (1992). An underlying trilineage dysplastic change in the bonemarrow cells of the patients is found in all subtypes. Information isavailable regarding the pathobiology of MDS, certain MDS classificationsystems, and particular methods of treating and managing MDS. See, e.g.,U.S. Pat. No. 7,189,740 (issued Mar. 13, 2007), which is incorporated byreference herein in its entirety.

Nucleoside analogs have been used clinically for the treatment of viralinfections and cancer. Most nucleoside analogs are classified asanti-metabolites. After they enter the cell, nucleoside analogs aresuccessively phosphorylated to nucleoside 5′-mono-phosphates,di-phosphates, and tri-phosphates.

5-Azacytidine (National Service Center designation NSC-102816; CASRegistry Number 320-67-2), also known as azacitidine, AZA, or4-amino-1-β-D-ribofuranosyl-1,3,5-triazin-2(1H)-one, is currentlymarketed as the drug product VIDAZA®. 5-Azacytidine is a nucleosideanalog, more specifically a cytidine analog. 5-Azacytidine is anantagonist of its related natural nucleoside, cytidine. 5-Azacytidineand 5-aza-2′-deoxycytidine (also known as decitabine, an analog ofdeoxycytidine) are also antagonists of deoxycytidine. A structuraldifference between these cytidine analogs and their related naturalnucleoside is the presence of a nitrogen at position 5 of the cytosinering in place of a carbon. 5-Azacytidine may be defined as having themolecular formula C₈H₁₂N₄O₅, a molecular weight of 244.21 grams permole, and the following structure:

Other members of the class of cytidine analogs include, for example:1-β-D-arabinofuranosylcytosine (Cytarabine or ara-C);5-aza-2′-deoxycytidine (Decitabine or 5-aza-CdR); pseudoisocytidine (psiICR); 5-fluoro-2′-deoxycytidine (FCdR); 2′-deoxy-2′,2′-difluorocytidine(Gemcitabine); 5-aza-2′-deoxy-2′,2′-difluorocytidine;5-aza-2′-deoxy-2′-fluorocytidine; 1-β-D-ribofuranosyl-2(1H)-pyrimidinone(Zebularine); 2′,3′-dideoxy-5-fluoro-3′-thiacytidine (Emtriva);2′-cyclocytidine (Ancitabine); 1-β-D-arabinofuranosyl-5-azacytosine(Fazarabine or ara-AC); 6-azacytidine (6-aza-CR);5,6-dihydro-5-azacytidine (dH-aza-CR);N⁴-pentyloxycarbonyl-5′-deoxy-5-fluorocytidine (Capecitabine);N⁴-octadecyl-cytarabine; and elaidic acid cytarabine.

After its incorporation into replicating DNA, 5-azacytidine or5-aza-2′-deoxycytidine forms a covalent complex with DNAmethyltransferases. DNA methyltransferases are responsible for de novoDNA methylation and for reproducing established methylation patterns indaughter DNA strands of replicating DNA. Inhibition of DNAmethyltransferases by 5-azacytidine or 5-aza-2′-deoxycytidine leads toDNA hypomethylation, thereby restoring normal functions tomorphologically dysplastic, immature hematopoietic cells and cancercells by re-expression of genes involved in normal cell cycleregulation, differentiation and death. The cytotoxic effects of thesecytidine analogs cause the death of rapidly dividing cells, includingcancer cells, that are no longer responsive to normal cell growthcontrol mechanisms. 5-azacytidine, unlike 5-aza-2′-deoxycytidine, alsoincorporates into RNA. The cytotoxic effects of azacitidine may resultfrom multiple mechanisms, including inhibition of DNA, RNA and proteinsynthesis, incorporation into RNA and DNA, and activation of DNA damagepathways.

5-Azacytidine and 5-aza-2′-deoxycytidine have been tested in clinicaltrials and showed significant anti-tumor activity, such as, for example,in the treatment of myelodysplastic syndromes (MDS), acute myelogenousleukemia (AML), chronic myelogenous leukemia (CIVIL), acute lymphocyticleukemia (ALL), and non Hodgkin's lymphoma (NHL). See, e.g., Aparicio etal., Curr. Opin. Invest. Drugs 3(4): 627-33 (2002). 5-Azacytidine hasundergone NCI-sponsored trials for the treatment of MDS and has beenapproved for treating all FAB subtypes of MDS. See, e.g., Kornblith etal., J. Clin. Oncol. 20(10): 2441-2452 (2002); Silverman et al., J.Clin. Oncol. 20(10): 2429-2440 (2002). 5-Azacytidine may alter thenatural course of MDS by diminishing the transformation to AML throughits cytotoxic activity and its inhibition of DNA methyltransferase. In aPhase III study, 5-azacytidine administered subcutaneously significantlyprolonged survival and time to AML transformation or death in subjectswith higher-risk MDS. See, e.g., P. Fenaux et al., Lancet Oncol., 2009,10(3):223-32; Silverman et al., Blood 106(11): Abstract 2526 (2005).

5-Azacytidine and other cytidine analogs are approved for subcutaneous(SC) or intravenous (IV) administration to treat various proliferativedisorders. Oral dosing of cytidine analogs would be more desirable andconvenient for patients and doctors, e.g., by eliminating injection-sitereactions that may occur with SC administration and/or by permittingimproved patient compliance. However, oral delivery of cytidine analogshas proven difficult due to combinations of chemical instability,enzymatic instability, and/or poor permeability. For example, cytidineanalogs have been considered acid labile and unstable in the acidicgastric environment. Previous attempts to develop oral dosage forms ofcytidine analogs have required enteric coating of the drug core toprotect the active pharmaceutical ingredient (API) from what wasunderstood and accepted to be therapeutically unacceptable hydrolysis inthe stomach, such that the drug is preferably absorbed in specificregions of the lower gastrointestinal tract, such as the jejunum in thesmall intestine. See, e.g., Sands, et al., U.S. Patent Publication No.2004/0162263 (application. Ser. No. 10/698,983). In addition, agenerally accepted belief in the art has been that water leads todetrimental hydrolytic degradation of cytidine analogs duringformulation, subsequently affecting the stability of the API in thedosage form. As a result, coatings applied to the drug core forprospective oral delivery of cytidine analogs have previously beenlimited to organic solvent-based systems to minimize exposure of the APIto water.

A great need remains for oral formulations and dosage forms of cytidineanalogs, such as, e.g., 5-azacytidine, to potentially permit, interalia, more advantageous dosing amounts or dosing periods; improvedpharmacokinetic profiles, pharmacodynamic profiles, or safety profiles;evaluation of the benefits of long-term or maintenance therapies;development of improved treatment regimens that maximize biologicactivity; use of cytidine analogs for treating new diseases ordisorders; and/or other potential advantageous benefits.

IV. SUMMARY

Provided herein are pharmaceutical compositions comprising cytidineanalogs, wherein the compositions release the API substantially in thestomach upon oral administration. Also provided are methods for makingthe compositions, and methods for using the compositions to treatdiseases and disorders including cancer, disorders related to abnormalcell proliferation, and hematologic disorders, among others.

In certain embodiments, the cytidine analog is 5-azacytidine. In otherembodiments, the cytidine analog is 5-aza-2′-deoxycytidine (decitabineor 5-aza-CdR). In yet other embodiments, the cytidine analog is, forexample: 1-β-D-arabinofuranosylcytosine (Cytarabine or ara-C);pseudoisocytidine (psi ICR); 5-fluoro-2′-deoxycytidine (FCdR);2′-deoxy-2′,2′-difluorocytidine (Gemcitabine);5-aza-2′-deoxy-2′,2′-difluorocytidine; 5-aza-2′-deoxy-2′-fluorocytidine;1-β-D-ribofuranosyl-2(1H)-pyrimidinone (Zebularine);2′,3′-dideoxy-5-fluoro-3′-thiacytidine (Emtriva); 2′-cyclocytidine(Ancitabine); 1-3-D-arabinofuranosyl-5-azacytosine (Fazarabine orara-AC); 6-azacytidine (6-aza-CR); 5,6-dihydro-5-azacytidine(dH-aza-CR); N⁴-pentyloxycarbonyl-5′-deoxy-5-fluorocytidine(Capecitabine); N⁴-octadecyl-cytarabine; elaidic acid cytarabine; ortheir derivatives or related analogs.

Certain embodiments herein provide compositions that are single unitdosage forms comprising a cytidine analog. Certain embodiments hereinprovide compositions that are non-enteric-coated. Certain embodimentsherein provide compositions that are tablets comprising a cytidineanalog. Certain embodiments herein provide compositions that arecapsules comprising a cytidine analog. The capsules may be, e.g., a hardgelatin capsule or a soft gelatin capsule; particular embodimentsprovide hydroxypropyl methylcellulose (HPMC) capsules. In certainembodiments, the single unit dosage forms optionally further contain oneor more excipients. In certain embodiments, the tablets optionallyfurther contain one or more excipients. In other embodiments, thecapsules optionally further contain one or more excipients. In certainembodiments, the composition is a tablet that effects an immediaterelease of the API upon oral administration. In other embodiments, thecomposition is a tablet that effects a controlled release of the APIsubstantially in the stomach. In certain embodiments, the composition isa capsule that effects an immediate release of the API upon oraladministration. In other embodiments, the composition is a capsule thateffects a controlled release of the API substantially in the stomach. Inparticular embodiments, the tablet contains a drug core that comprises acytidine analog, and optionally further contains a coating of the drugcore, wherein the coating is applied to the drug core using an aqueoussolvent, such as, for example, water, or non-aqueous solvent, such as,for example ethanol.

Certain embodiments herein provide methods of making formulations ofcytidine analogs intended for oral delivery. Further provided arearticles of manufacture containing packaging material, an oralformulation of a cytidine analog, and a label that indicates that theformulation is for the treatment of certain diseases or disordersincluding, e.g., a cancer, a disorder related to abnormal cellproliferation, a hematologic disorder, or an immune disorder.

Certain embodiments herein provide methods of using the formulationsprovided herein to treat diseases or disorders including, e.g., cancer,disorders related to abnormal cell proliferation, hematologic disorders,or immune disorders, among others. In certain embodiments, theformulations of cytidine analogs are orally administered to subjects inneed thereof to treat a cancer or a hematological disorder, such as, forexample, MDS, AML, ALL, CML, NHL, leukemia, or lymphoma; or a solidtumor, such as, for example, sarcoma, melanoma, carcinoma, or cancer ofthe colon, breast, ovary, gastrointestinal system, kidney, lung (e.g.,non-small-cell lung cancer and small-cell lung cancer), testicle,prostate, pancreas or bone. In certain embodiments, the formulations ofcytidine analogs are orally administered to subjects in need thereof totreat an immune disorder. In certain embodiments, the oral formulationsprovided herein are co-administered with one or more therapeutic agentsto provide a synergistic therapeutic effect in subjects in need thereof.In certain embodiments, the oral formulations provided herein areco-administered with one or more therapeutic agents to provide aresensitization effect in subjects in need thereof. The co-administeredagents may be a cancer therapeutic agent, as described herein. Incertain embodiments, the co-administered agent(s) may be dosed, e.g.,orally or by injection.

In particular embodiments, provided herein are tablets containing5-azacytidine and methods for making and using the tablets to treatcancer, disorders related to abnormal cell proliferation, or hematologicdisorders. In certain embodiments, the tablets optionally furthercontain one or more excipients such as, for example, glidants, diluents,lubricants, colorants, disintegrants, granulating agents, bindingagents, polymers, and/or coating agents. Examples of ingredients usefulin preparing certain formulations provided herein are described in,e.g., Etter et al., U.S. Patent Application Publication No. 2008/0057086(application. Ser. No. 11/849,958), which is incorporated herein byreference in its entirety.

Specific embodiments herein provide, inter alia, pharmaceuticalcompositions comprising a therapeutically effective amount of5-azacytidine, wherein the composition releases the 5-azacytidinesubstantially in the stomach following oral administration to a subject.Further embodiments provide the aforementioned compositions, which: areimmediate release compositions; do not have an enteric coating (i.e.,are non-enteric-coated); are tablets; are capsules; further comprise anexcipient selected from any excipient disclosed herein; further comprisea permeation enhancer; further comprise d-alpha-tocopheryl polyethyleneglycol 1000 succinate; further comprise a permeation enhancer in theformulation at about 2% by weight relative to the total weight of theformulation; are essentially free of a cytidine deaminase inhibitor; areessentially free of tetrahydrouridine; have an amount of 5-azacytidineof at least about 40 mg; have an amount of 5-azacytidine of at leastabout 400 mg; have an amount of 5-azacytidine of at least about 1000 mg;achieve an area-under-the-curve value of at least about 200 ng-hr/mLfollowing oral administration to a subject; achieve anarea-under-the-curve value of at least about 400 ng-hr/mL following oraladministration to a subject; achieve a maximum plasma concentration ofat least about 100 ng/mL following oral administration to a subject;achieve a maximum plasma concentration of at least about 200 ng/mLfollowing oral administration to a subject; achieve a time to maximumplasma concentration of less than about 90 minutes following oraladministration to a subject; and/or achieve a time to maximum plasmaconcentration of less than about 60 minutes following oraladministration to a subject.

Specific embodiments herein provide a pharmaceutical composition fororal administration comprising a therapeutically effective amount of5-azacytidine, which releases the 5-azacytidine substantially in thestomach and achieves an area-under-the-curve value of at least about 200ng-hr/mL following oral administration.

Specific embodiments herein provide a pharmaceutical composition fororal administration comprising a therapeutically effective amount of5-azacytidine, which releases the 5-azacytidine substantially in thestomach and achieves an area-under-the-curve value of at least about 400ng-hr/mL following oral administration.

Specific embodiments herein provide a pharmaceutical composition fororal administration comprising a therapeutically effective amount of5-azacytidine, which releases the 5-azacytidine substantially in thestomach and achieves a maximum plasma concentration of at least about100 ng/mL following oral administration.

Specific embodiments herein provide a pharmaceutical composition fororal administration comprising a therapeutically effective amount of5-azacytidine, which releases the 5-azacytidine substantially in thestomach and achieves a maximum plasma concentration of at least about200 ng/mL following oral administration.

Specific embodiments herein provide a pharmaceutical composition fororal administration comprising a therapeutically effective amount of5-azacytidine, which releases the 5-azacytidine substantially in thestomach and achieves a time to maximum plasma concentration of, e.g.,less than about 6 hr, less than about 5 hr, less than about 4 hr, lessthan about 3 hr, less than about 2.5 hr, less than about 2 hr, less thanabout 1.5 hr, less than about 1 hr, less than about 45 min, or less thanabout 30 min following oral administration. In specific embodiments, thepresence of food may affect (e.g., extend) the total exposure and/ortime to maximum plasma concentration.

Specific embodiments herein provide a pharmaceutical composition fororal administration comprising a therapeutically effective amount of5-azacytidine, which releases the 5-azacytidine substantially in thestomach and achieves a time to maximum plasma concentration of less thanabout 60 minutes following oral administration.

Specific embodiments herein provide any of the aforementionedcompositions, as single unit dosage forms, tablets, or capsules.

Specific embodiments herein provide, inter alia, methods for treating asubject having a disease associated with abnormal cell proliferation,comprising orally administering to the subject a pharmaceuticalcomposition comprising a therapeutically effective amount of5-azacytidine, wherein the composition releases the 5-azacytidinesubstantially in the stomach following oral administration to thesubject. Further embodiments herein provide the aforementioned methods,in which: the disease is myelodysplastic syndrome; the disease is acutemyelogenous leukemia; the method further comprises co-administering tothe subject in need thereof an additional therapeutic agent selectedfrom any additional therapeutic agent disclosed herein; the compositionis an immediate release composition; the composition does not have anenteric coating; the composition further comprises a permeationenhancer; the composition further comprises the permeation enhancerd-alpha-tocopheryl polyethylene glycol 1000 succinate; the compositionfurther comprises d-alpha-tocopheryl polyethylene glycol 1000 succinatein the formulation at about 2% by weight relative to the total weight ofthe formulation; the method further comprises not co-administering acytidine deaminase inhibitor with the cytidine analog; the compositionis a single unit dosage form; the composition is a tablet; thecomposition is a capsule; the composition further comprises an excipientselected from any excipient disclosed herein; the amount of5-azacytidine is at least about 40 mg; the amount of 5-azacytidine is atleast about 400 mg; the amount of 5-azacytidine is at least about 1000mg; the method achieves an area-under-the-curve value of at least about200 ng-hr/mL following oral administration to the subject; the methodachieves an area-under-the-curve value of at least about 400 ng-hr/mLfollowing oral administration to the subject; the method achieves amaximum plasma concentration of at least about 100 ng/mL following oraladministration to the subject; the method achieves a maximum plasmaconcentration of at least about 200 ng/mL following oral administrationto the subject; the method achieves a time to maximum plasmaconcentration of less than about 90 minutes following oraladministration to the subject; and/or the method achieves a time tomaximum plasma concentration of less than about 60 minutes followingoral administration to the subject

Specific embodiments herein provide, inter alia, pharmaceuticalcompositions comprising a therapeutically effective amount of5-azacytidine, wherein the compositions are for treating a disease ordisorder associated with abnormal cell proliferation, wherein thecompositions are prepared for oral administration, and wherein thecompositions are prepared for release of the 5-azacytidine substantiallyin the stomach. Further embodiments herein provide the aforementionedcompositions, which: have an amount of 5-azacytidine of about 40 mg,about 400 mg, or about 1000 mg; are prepared to achieve anarea-under-the-curve value of at least about 200 ng-hr/mL or 400ng-hr/mL following oral administration; are prepared to achieve amaximum plasma concentration of at least about 100 ng/mL or 200 ng/mLfollowing oral administration; are prepared to achieve a time to maximumplasma concentration of less than about 60 minutes or 90 minutes afterbeing administered; are prepared in the form of an immediate releasecomposition; are prepared for oral administration in combination with anadditional therapeutic agent selected from any additional therapeuticagent disclosed herein; are for treating myelodysplastic syndrome oracute myelogenous leukemia; further comprise a permeation enhancer;which further comprise the permeation enhancer d-alpha-tocopherylpolyethylene glycol 1000 succinate; are single unit dosage forms; aretablets or capsules; and/or further comprise an excipient selected fromany excipient disclosed herein.

Specific embodiments herein provide, inter alia, uses of 5-azacytidinefor the preparation of a pharmaceutical composition for treating adisease associated with abnormal cell proliferation, wherein thecomposition is prepared for oral administration, and wherein thecomposition is prepared for release of the 5-azacytidine substantiallyin the stomach. Further embodiments herein provide the aforementioneduses, in which: the disease is myelodysplastic syndrome or acutemyelogenous leukemia; the amount of 5-azacytidine is selected from anyamount disclosed herein; and/or the composition is prepared forimmediate release. Further embodiments provide, inter alia, methods fortreating a subject having a disease or disorder provided herein byadministering a pharmaceutical compositions provided herein, wherein thetreatment results in improved survival of the subject.

Provided herein is a method of treating diffuse large B-cell lymphoma,follicular lymphoma, or mantel cell lymphoma, which comprisesadministering to a human having diffuse large B-cell lymphoma,follicular lymphoma, or mantel cell lymphoma a therapeutically effectiveamount of 5-azacytidine, or a pharmaceutically acceptable salt, solvateor hydrate thereof, and optionally administering therapeuticallyeffective amounts of one or more additional active agents.

Specific embodiments herein provide, inter alia, methods for treatingdiffuse large B-cell lymphoma, follicular lymphoma, or mantel celllymphoma by administering therapeutically effective amounts of5-azacytidine and one or more additional active agents comprisingrituximab, cyclophosphamide, doxorubicin, vincristine, prednisone, or acombination thereof. Further embodiments herein provide methods fortreating diffuse large B-cell lymphoma, follicular lymphoma, or mantelcell lymphoma wherein the 5-azacytidine and the one or more additionalactive agents are administered cyclically.

V. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents processes and steps that may be used to makeparticular tablets comprising azacitidine for oral dosing; in specificembodiments, one or more steps may be optionally omitted.

FIG. 2 represents human PK profiles following 75 mg/m² SC dosing ofazacitidine on Days 1 and 7 in a multiple dose escalation study (n=18).The X-axis represents time; the Y-axis represents azacitidine plasmaconcentrations (mean±SD).

FIG. 3 represents human PK profiles following SC (75 mg/m²) and PO (240mg, 300 mg, and 360 mg) dosing of azacitidine in a multiple doseescalation study. The azacitidine plasma PK profiles are compared amongvarious doses. The X-axis represents time; the Y-axis representsazacitidine plasma concentrations (mean±SD).

FIGS. 4A-4D represent PD data from an individual patient (Subject 02008,80 year old white male, RAEB-1) collected during a multiple doseescalation study. The patient was dosed with azacitidine Formulation #3,240 mg. Platelets (K/μL), Hgb (g/dL), ANC (K/μL), and Relative BM Blast(%) are plotted versus sampling dates over the course of the study.

FIGS. 5A-5D represent PD data from an individual patient (Subject 02007,76 year old white male, CMML) collected during a multiple doseescalation study. The patient was dosed with azacitidine Formulation #3,240 mg. Platelets (K/μL), Hgb (g/dL), ANC (K/μL), and Relative BM Blast(%) are plotted versus sampling dates over the course of the study.

FIGS. 6A-6D represent PD data from an individual patient (Subject 02004,61 year old white male, MDS, MDACC) collected during a multiple doseescalation study. The patient was dosed with azacitidine Formulation 1,120 mg. Platelets (K/μL), Hgb (g/dL), ANC (K/μL), and Relative BM Blast(%) are plotted versus sampling dates over the course of the study.

FIG. 7 represents a study design of a Rapid Aza Clinical Evaluation(RACE) study CL008. Doses given on various days within a treatment cycleare depicted. Dose may be administered±1 day, as long as there is atleast 48 hours between doses.

FIG. 8 represents azacitidine human PK profiles from an individualpatient (Subject 106003, N=1) following SC (124 mg, 75 mg/m²) and PO(180 mg, 360 mg, 1,200 mg, Formulation 4) dosing of azacitidine from aRACE clinical study. AUC(0-t) values for the SC and PO doses aredepicted.

FIG. 9 represents azacitidine human PK profiles from an individualpatient (Subject 106004, N=1) following SC (120 mg, 75 mg/m²) and PO(180 mg, 360 mg, 1,200 mg, Formulation 6) dosing of azacitidine from aRACE clinical study. AUC(0-∞) values for the SC and PO doses aredepicted.

FIG. 10 represents human PK profiles (linear scale) following SC andoral administration of azacitidine in clinical studies.

FIG. 11 represents human PK profiles (semi-log scale) following SC andoral administration of azacitidine in clinical studies.

FIG. 12 represents human AUC values following SC dosing of azacitidineand oral dosing of azacitidine with Formulations #3, #4, and #6 atvarious dosage levels in clinical studies (CL005 and CL008).

FIG. 13 represents human Cmax values in patients following SC dosing ofazacitidine and oral dosing of azacitidine with Formulations #3, #4, and#6 at various dosage levels in clinical studies.

FIG. 14 represents relative oral bioavailability in humans followingoral dosing of azacitidine with Formulations #3, #4, and #6 at variousdosage levels.

FIG. 15 represents percent exposure in humans relative to SCadministration following oral dosing of azacitidine with Formulations#3, #4, and #6 at various dosage levels.

FIG. 16 represents profiles of human plasma concentration versus time(linear scale) following oral dosing of azacitidine with Formulations #3and #6 and 180 mg (n=6).

FIG. 17 represents linear scale profiles of human plasma concentration(ng/ml) versus time (hr) following oral dosing of azacitidine withFormulations #3 and #6 and 360 mg (n=6).

FIG. 18 represents a plot of values for individual (“ind”) and meanazacitidine ACU(0-inf) (ng*hr/ml) versus azacitidine dose (mg), withcalculated linear regression lines for Formulations #3 and #6.

FIG. 19 represents a comparison of azacitidine relative oralbioavailability (%) (mean±SD) versus azacitidine dose (mg) followingdosing with Formulation #3 or #6.

FIG. 20 represents a comparison of azacitidine exposure as compared toSC dose (mean±SD) versus azacitidine dose (mg) following oraladministration of Formulation #3 or #6.

VI. DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. All publications and patents referred to herein areincorporated by reference herein in their entireties.

A. Definitions

As used in the specification and the accompanying claims, the indefinitearticles “a” and “an” and the definite article “the” include plural aswell as singular referents, unless the context clearly dictatesotherwise.

The term “about” or “approximately” means an acceptable error for aparticular value as determined by one of ordinary skill in the art,which depends in part on how the value is measured or determined. Incertain embodiments, the term “about” or “approximately” means within 1,2, 3, or 4 standard deviations. In certain embodiments, the term “about”or “approximately” means within 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%,5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, or 0.05% of a given value or range.

As used herein, and unless otherwise specified, the terms “treat,”“treating” and “treatment” refer to the eradication or amelioration of adisease or disorder, or of one or more symptoms associated with thedisease or disorder. In certain embodiments, the terms refer tominimizing the spread or worsening of the disease or disorder resultingfrom the administration of one or more prophylactic or therapeuticagents to a subject with such a disease or disorder. In someembodiments, the terms refer to the administration of a compound ordosage form provided herein, with or without one or more additionalactive agent(s), after the onset of symptoms of the particular disease.

As used herein, and unless otherwise specified, the terms “prevent,”“preventing” and “prevention” refer to the prevention of the onset,recurrence or spread of a disease or disorder, or of one or moresymptoms thereof. In certain embodiments, the terms refer to thetreatment with or administration of a compound or dosage form providedherein, with or without one or more other additional active agent(s),prior to the onset of symptoms, particularly to subjects at risk ofdisease or disorders provided herein. The terms encompass the inhibitionor reduction of a symptom of the particular disease. Subjects withfamilial history of a disease in particular are candidates forpreventive regimens in certain embodiments. In addition, subjects whohave a history of recurring symptoms are also potential candidates forprevention. In this regard, the term “prevention” may be interchangeablyused with the term “prophylactic treatment.”

As used herein, and unless otherwise specified, the terms “manage,”“managing” and “management” refer to preventing or slowing theprogression, spread or worsening of a disease or disorder, or of one ormore symptoms thereof. Often, the beneficial effects that a subjectderives from a prophylactic and/or therapeutic agent do not result in acure of the disease or disorder. In this regard, the term “managing”encompasses treating a subject who had suffered from the particulardisease in an attempt to prevent or minimize the recurrence of thedisease.

As used herein, amelioration of the symptoms of a particular disorder byadministration of a particular pharmaceutical composition refers to anylessening, whether permanent or temporary, lasting or transient, thatcan be attributed to or associated with administration of thecomposition.

As used herein, and unless otherwise specified, the terms“therapeutically effective amount” and “effective amount” of a compoundmean an amount sufficient to provide a therapeutic benefit in thetreatment or management of a disease or disorder, or to delay orminimize one or more symptoms associated with the disease or disorder. A“therapeutically effective amount” and “effective amount” of a compoundmean an amount of therapeutic agent, alone or in combination with one ormore other agent(s), which provides a therapeutic benefit in thetreatment or management of the disease or disorder. The terms“therapeutically effective amount” and “effective amount” can encompassan amount that improves overall therapy, reduces or avoids symptoms orcauses of disease or disorder, or enhances the therapeutic efficacy ofanother therapeutic agent.

As used herein, and unless otherwise specified, a “prophylacticallyeffective amount” of a compound is an amount sufficient to prevent adisease or disorder, or prevent its recurrence. A prophylacticallyeffective amount of a compound means an amount of therapeutic agent,alone or in combination with one or more other agent(s), which providesa prophylactic benefit in the prevention of the disease. The term“prophylactically effective amount” can encompass an amount thatimproves overall prophylaxis or enhances the prophylactic efficacy ofanother prophylactic agent.

“Tumor,” as used herein, refers to all neoplastic cell growth andproliferation, whether malignant or benign, and all pre-cancerous andcancerous cells and tissues. “Neoplastic,” as used herein, refers to anyform of dysregulated or unregulated cell growth, whether malignant orbenign, resulting in abnormal tissue growth. Thus, “neoplastic cells”include malignant and benign cells having dysregulated or unregulatedcell growth.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include, but are not limitedto blood borne (e.g., lymphoma, leukemia) and solid tumors.

The terms “composition,” “formulation,” and “dosage form,” as usedherein are intended to encompass compositions comprising the specifiedingredient(s) (in the specified amounts, if indicated), as well as anyproduct(s) which result, directly or indirectly, from combination of thespecified ingredient(s) in the specified amount(s). By “pharmaceutical”or “pharmaceutically acceptable” it is meant that any diluent(s),excipient(s) or carrier(s) in the composition, formulation, or dosageform are compatible with the other ingredient(s) and not deleterious tothe recipient thereof. Unless indicated otherwise, the terms“composition,” “formulation,” and “dosage form” are used hereininterchangeably.

The term “immediate release,” when used herein in reference to acomposition, formulation, or dosage form provided herein, means that thecomposition, formulation, or dosage form does not comprise a component(e.g., a coating) that serves to delay the spatial and/or temporalrelease of some or all of the API from the composition, formulation, ordosage form beyond the stomach following oral administration. In certainembodiments, an immediate release composition, formulation, or dosageform is one that releases the API substantially in the stomach followingoral administration. In specific embodiments, an immediate releasecomposition, formulation, or dosage form is one that is notdelayed-release. In specific embodiments, an immediate releasecomposition, formulation, or dosage form is one that does not comprisean enteric coating.

The term “non-enteric-coated,” when used herein, refers to apharmaceutical composition, formulation, or dosage form that does notcomprise a coating intended to release the active ingredient(s) beyondthe stomach (e.g., in the intestine). In certain embodiments, anon-enteric-coated composition, formulation, or dosage form is designedto release the active ingredient(s) substantially in the stomach.

The term “substantially in the stomach,” when used herein in referenceto a composition, formulation, or dosage form provided herein, meansthat at least about 99%, at least about 95%, at least about 90%, atleast about 85%, at least about 80%, at least about 75%, at least about70%, at least about 65%, at least about 60%, at least about 55%, atleast about 50%, at least about 45%, at least about 40%, at least about35%, at least about 30%, at least about 25%, at least about 20%, atleast about 15%, or at least about 10% of the cytidine analog isreleased in the stomach. The term “released in the stomach” and relatedterms as used herein refer to the process whereby the cytidine analog ismade available for uptake by or transport across cells lining thestomach and then made available to the body.

The term “subject” is defined herein to include animals such as mammals,including, but not limited to, primates (e.g., humans), cows, sheep,goats, horses, dogs, cats, rabbits, rats, mice and the like. In specificembodiments, the subject is a human.

The terms “co-administration” and “in combination with” include theadministration of two or more therapeutic agents either simultaneously,concurrently or sequentially within no specific time limits. In oneembodiment, the agents are present in the cell or in the subject's bodyat the same time or exert their biological or therapeutic effect at thesame time. In one embodiment, the therapeutic agents are in the samecomposition or unit dosage form. In other embodiments, the therapeuticagents are in separate compositions or unit dosage forms. In certainembodiments, a first agent can be administered prior to (e.g., 5minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before),concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks,5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of asecond therapeutic agent.

The term “isotopic composition” refers to the amount of each isotopepresent in a given atomic position, and “natural isotopic composition”refers to the naturally occurring isotopic composition or abundance fora given atomic position. Atomic positions containing their naturalisotopic composition may also be referred to herein as “non-enriched.”Unless otherwise designated, the atomic positions of the compoundsrecited herein are meant to represent any stable isotope of that atom.For example, unless otherwise stated, when a position is designatedspecifically as “H” or “hydrogen,” the position is understood to havehydrogen at its natural isotopic composition.

The term “isotopically enriched” refers to an atomic position having anisotopic composition other than the natural isotopic composition of thatatom. “Isotopically enriched” may also refer to a compound containing atleast one atomic position having an isotopic composition other than thenatural isotopic composition of that atom. As used herein, an“isotopologue” is an isotopically enriched compound.

The term “isotopic enrichment” refers to the percentage of incorporationof an amount of a specific isotope at a given atomic position in amolecule in the place of that atom's natural isotopic composition. Forexample, deuterium enrichment of 1% at a given position means that 1% ofthe molecules in a given sample contain deuterium at the specifiedposition. Because the naturally occurring distribution of deuterium isabout 0.0156%, deuterium enrichment at any position in a compoundsynthesized using non-enriched starting materials is about 0.0156%.

The term “isotopic enrichment factor” refers to the ratio between theisotopic composition and the natural isotopic composition of a specifiedisotope.

With regard to the compounds provided herein, when a particular atomicposition is designated as having deuterium or “D,” it is understood thatthe abundance of deuterium at that position is substantially greaterthan the natural abundance of deuterium, which is about 0.015%. Aposition designated as having deuterium typically has a minimum isotopicenrichment factor of, in particular embodiments, at least 1000 (15%deuterium incorporation), at least 2000 (30% deuterium incorporation),at least 3000 (45% deuterium incorporation), at least 3500 (52.5%deuterium incorporation), at least 4000 (60% deuterium incorporation),at least 4500 (67.5% deuterium incorporation), at least 5000 (75%deuterium incorporation), at least 5500 (82.5% deuterium incorporation),at least 6000 (90% deuterium incorporation), at least 6333.3 (95%deuterium incorporation), at least 6466.7 (97% deuterium incorporation),at least 6600 (99% deuterium incorporation), or at least 6633.3 (99.5%deuterium incorporation) at each designated deuterium position.

The isotopic enrichment and isotopic enrichment factor of the compoundsprovided herein can be determined using conventional analytical methodsknown to one of ordinary skill in the art, including, e.g., massspectrometry, nuclear magnetic resonance spectroscopy, andcrystallography.

B. Cytidine Analogs

1. Overview

Provided herein are dosage forms, pharmaceutical formulations andcompositions comprising cytidine analogs that release the APIsubstantially in the stomach upon oral administration. In certainembodiments, the cytidine analog is 5-azacytidine. In certainembodiments, the cytidine analog is 5-aza-2′-deoxycytidine (decitabineor 5-aza-CdR). In certain embodiments, the cytidine analog is, forexample: 1-β-D-arabinofuranosylcytosine (Cytarabine or ara-C);pseudoiso-cytidine (psi ICR); 5-fluoro-2′-deoxycytidine (FCdR);2′-deoxy-2′,2′-difluorocytidine (Gemcitabine);5-aza-2′-deoxy-2′,2′-difluorocytidine; 5-aza-2′-deoxy-2′-fluorocytidine;1-β-D-ribofuranosyl-2(1H)-pyrimidinone (Zebularine);2′,3′-dideoxy-5-fluoro-3′-thiacytidine (Emtriva); 2′-cyclocytidine(Ancitabine); 1-β-D-arabinofuranosyl-5-azacytosine (Fazarabine orara-AC); 6-azacytidine (6-aza-CR); 5,6-dihydro-5-azacytidine(dH-aza-CR); N⁴-pentyloxy-carbonyl-5′-deoxy-5-fluorocytidine(Capecitabine); N⁴-octadecyl-cytarabine; elaidic acid cytarabine; or aconjugated compound comprising a cytidine analog and a fatty acid (e.g.,an azacitidine-fatty acid conjugate, including, but not limited to,CP-4200 (Clavis Pharma ASA) or a compound disclosed in WO 2009/042767,such as aza-C-5′-petroselinic acid ester or aza-C-5′-petroselaidic acidester).

In certain embodiments, cytidine analogs provided herein includeesterified derivatives of cytidine analogs, such as, e.g., esterifiedderivatives of 5-azacytidine. In particular embodiments, esterifiedderivatives are cytidine analogs that contain an ester moiety (e.g., anacetyl group) at one or more positions on the cytidine analog molecule.Esterified derivatives may be prepared by any method known in the art.In certain embodiments, esterified derivatives of a cytidine analogserve as prodrugs of the cytidine analog, such that, e.g., followingadministration of an esterified derivative, the derivative isdeacetylated in vivo to yield the cytidine analog. A particularembodiment herein provides 2′,3′,5′-triacetyl-5-azacytidine (TAC), whichpossesses favorable physical-chemical and therapeutic properties. See,e.g., International Publication No. WO 2008/092127 (InternationalApplication No. PCT/US2008/052124); Ziemba, A. J., et al., “Developmentof Oral Demethylating Agents for the Treatment of MyelodysplasticSyndrome” (Abstract No. 3369), In: Proceedings of the 100th AnnualMeeting of the American Association for Cancer Research; 2009 Apr.18-22; Denver, Co. Philadelphia (Pa.): AACR; 2009 (both of which areincorporated by reference herein in their entireties).

In certain embodiments, the cytidine analogs provided herein include anycompound which is structurally related to cytidine or deoxycytidine andfunctionally mimics and/or antagonizes the action of cytidine ordeoxycytidine. Certain embodiments herein provide salts, cocrystals,solvates (e.g., hydrates), complexes, prodrugs, precursors, metabolites,and/or other derivatives of the cytidine analogs provided herein. Forexample, particular embodiments provide salts, cocrystals, solvates(e.g., hydrates), complexes, precursors, metabolites, and/or otherderivatives of 5-azacytidine. Certain embodiments provide cytidineanalogs that are not salts, cocrystals, solvates (e.g., hydrates), orcomplexes of the cytidine analogs provided herein. For example,particular embodiments provide 5-azacytidine in a non-ionized,non-solvated (e.g., anhydrous), non-complexed form. Certain embodimentsherein provide mixtures of two or more cytidine analogs provided herein.

Cytidine analogs provided herein may be prepared using synthetic methodsand procedures referenced herein or otherwise available in theliterature. For example, particular methods for synthesizing5-azacytidine are taught in, e.g., U.S. Pat. No. 7,038,038 andreferences discussed therein, each of which is incorporated herein byreference. 5-Azacytidine is also available from Celgene Corporation,Warren, N.J. Other cytidine analogs provided herein may be preparedusing previously disclosed synthetic procedures available to a person ofordinary skill in the art.

In certain embodiments, exemplary cytidine analogs have the structuresprovided below:

2. Isotopically Enriched Cytidine Analogs

Particular embodiments herein provide isotopically enriched cytidineanalogs, prodrugs thereof, synthetic intermediates thereof, andmetabolites thereof. For example, specific embodiments herein provideisotopically enriched 5-azacytidine.

Isotopic enrichment (e.g., deuteration) of pharmaceuticals to improvepharmacokinetics (“PK”), pharmacodynamics (“PD”), and toxicity profiles,has been demonstrated previously with some classes of drugs. See, e.g.,Lijinsky et. al., Food Cosmet. Toxicol., 20: 393 (1982); Lijinsky et.al., J. Nat. Cancer Inst., 69: 1127 (1982); Mangold et. al., MutationRes. 308: 33 (1994); Gordon et. al., Drug Metab. Dispos., 15: 589(1987); Zello et. al., Metabolism, 43: 487 (1994); Gately et. al., J.Nucl. Med., 27: 388 (1986); Wade, D., Chem. Biol. Interact. 117: 191(1999).

Without being limited by any particular theory, isotopic enrichment of adrug can be used, for example, to: (1) reduce or eliminate unwantedmetabolites; (2) increase the half-life of the parent drug; (3) decreasethe number of doses needed to achieve a desired effect; (4) decrease theamount of a dose necessary to achieve a desired effect; (5) increase theformation of active metabolites, if any are formed; and/or (6) decreasethe production of deleterious metabolites in specific tissues and/orcreate a more effective drug and/or a safer drug for combinationtherapy, whether the combination therapy is intentional or not.

Replacement of an atom for one of its isotopes may often result in achange in the reaction rate of a chemical reaction. This phenomenon isknown as the Kinetic Isotope Effect (“KIE”). For example, if a C—H bondis broken during a rate-determining step in a chemical reaction (i.e.the step with the highest transition state energy), substitution of adeuterium for that hydrogen will cause a decrease in the reaction rateand the process will slow down. This phenomenon is known as theDeuterium Kinetic Isotope Effect (“DKIE”). See, e.g, Foster et al., Adv.Drug Res., vol. 14, pp. 1-36 (1985); Kushner et al., Can. J. Physiol.Pharmacol., vol. 77, pp. 79-88 (1999).

The magnitude of the DKIE can be expressed as the ratio between therates of a given reaction in which a C—H bond is broken, and the samereaction where deuterium is substituted for hydrogen. The DKIE can rangefrom about 1 (no isotope effect) to very large numbers, such as 50 ormore, meaning that the reaction can be fifty, or more, times slower whendeuterium is substituted for hydrogen. Without being limited by aparticular theory, high DKIE values may be due in part to a phenomenonknown as tunneling, which is a consequence of the uncertainty principle.Tunneling is ascribed to the small mass of a hydrogen atom, and occursbecause transition states involving a proton can sometimes form in theabsence of the required activation energy. Because deuterium has moremass than hydrogen, it statistically has a much lower probability ofundergoing this phenomenon.

Tritium (“T”) is a radioactive isotope of hydrogen, used in research,fusion reactors, neutron generators and radiopharmaceuticals. Tritium isa hydrogen atom that has 2 neutrons in the nucleus and has an atomicweight close to 3. It occurs naturally in the environment in very lowconcentrations, most commonly found as T₂O. Tritium decays slowly(half-life=12.3 years) and emits a low energy beta particle that cannotpenetrate the outer layer of human skin. Internal exposure is the mainhazard associated with this isotope, yet it must be ingested in largeamounts to pose a significant health risk. As compared with deuterium, alesser amount of tritium must be consumed before it reaches a hazardouslevel. Substitution of tritium (“T”) for hydrogen results in yet astronger bond than deuterium and gives numerically larger isotopeeffects.

Similarly, substitution of isotopes for other elements, including, butnot limited to, ¹³C or ¹⁴C for carbon, ³³S, ³⁴S, or ³⁶S for sulfur, ¹⁵Nfor nitrogen, and ¹⁷O or ¹⁸O for oxygen, may lead to an analogouskinetic isotope effect.

The animal body expresses a variety of enzymes for the purpose ofeliminating foreign substances, such as therapeutic agents, from itscirculation system. Examples of such enzymes include the cytochrome P450enzymes (“CYPs”), esterases, proteases, reductases, dehydrogenases, andmonoamine oxidases, to react with and convert these foreign substancesto more polar intermediates or metabolites for renal excretion. Some ofthe most common metabolic reactions of pharmaceutical compounds involvethe oxidation of a carbon-hydrogen (C—H) bond to either a carbon-oxygen(C—O) or carbon-carbon (C—C) pi-bond. The resultant metabolites may bestable or unstable under physiological conditions, and can havesubstantially different pharmacokinetic, pharmacodynamic, and acute andlong-term toxicity profiles relative to the parent compounds. For manydrugs, such oxidations are rapid. As a result, these drugs often requirethe administration of multiple or high daily doses.

Isotopic enrichment at certain positions of a compound provided hereinmay produce a detectable KIE that affects the pharmacokinetic,pharmacologic, and/or toxicological profiles of a compound providedherein in comparison with a similar compound having a natural isotopiccomposition. In one embodiment, the deuterium enrichment is performed onthe site of C—H bond cleavage during metabolism.

Certain embodiments herein provide deuterium enriched 5-azacytidineanalogs, wherein one or more hydrogen(s) in the 5-azacytidine moleculeis/are isotopically enriched with deuterium. In certain embodiments,provided herein are compounds of formula (I):

wherein one or more Y atom(s) (i.e., Y¹, Y², Y³, Y⁴, Y⁵, Y⁶, and Y⁷)is/are hydrogen(s) isotopically enriched with deuterium, and anyremaining Y atom(s) is/are non-enriched hydrogen atom(s). In particularembodiments, one, two, three, four, five, six, or seven of the indicatedY atom(s) is/are isotopically enriched with deuterium, and any remainingY atom(s) is/are non-enriched hydrogen(s).

In certain embodiments, one or more Y atoms on the ribose moiety ofCompound (I) are deuterium-enriched. Particular examples include, butare not limited to, the following compounds, in which the label “D”indicates a deuterium-enriched atomic position, i.e., a samplecomprising the given compound has a deuterium enrichment at theindicated position(s) above the natural abundance of deuterium:

In certain embodiments, the Y atom on the 5-azacytosine moiety ofCompound (I) is deuterium-enriched. Particular example includes thefollowing compound, in which the label “D” indicates adeuterium-enriched atomic position, i.e., a sample comprising the givencompound has a deuterium enrichment at the indicated position(s) abovethe natural abundance of deuterium:

In certain embodiments, one or more Y atoms on the ribose moiety and theY atom on the 5-azacytosine moiety of Compound (I) aredeuterium-enriched. Particular examples include, but are not limited to,the following compounds, in which the label “D” indicates adeuterium-enriched atomic position, i.e., a sample comprising the givencompound has a deuterium enrichment at the indicated position(s) abovethe natural abundance of deuterium:

It is understood that one or more deuterium(s) may exchange withhydrogen under physiological conditions.

Certain embodiments herein provide carbon-13 enriched analogs of5-azacytidine, wherein one or more carbon(s) in the 5-azacytidinemolecule is/are isotopically enriched with carbon-13. In certainembodiments, provided herein are compounds of formula (II):

wherein one or more of 1, 2, 3, 4, 5, 6, 7, or 8 is/are carbon atom(s)isotopically enriched with carbon-13, and any remaining atom(s) of 1, 2,3, 4, 5, 6, 7, or 8 is/are non-enriched carbon atom(s). In particularembodiments, one, two, three, four, five, six, seven, or eight carbonatom(s) (i.e., atoms 1, 2, 3, 4, 5, 6, 7, and 8) is/are isotopicallyenriched with carbon-13, and any remaining carbon atom(s) is/arenon-enriched.

In certain embodiments, one or more carbon atom(s) of the ribose moietyof Compound (II) are enriched with carbon-13. Particular examplesinclude, but are not limited to, the following compounds, in which theasterisk (“*”) indicates a carbon-13 enriched atomic position, i.e., asample comprising the given compound has a carbon-13 enrichment at theindicated position(s) above the natural abundance of carbon-13:

In certain embodiments, one or more carbon atom(s) of the 5-azacytosinemoiety of Compound (II) are enriched with carbon-13. Particular examplesinclude, but are not limited to, the following compounds, in which theasterisk “*” indicates a carbon-13 enriched atomic position, i.e., asample comprising the given compound has a carbon-13 enrichment at theindicated position(s) above the natural abundance of carbon-13:

In certain embodiments, one or more carbon atoms on the ribose moietyand one or more carbon atoms on the 5-azacytosine moiety of Compound(II) are enriched with carbon-13, i.e., any combination of carbon-13enrichment for the ribose moiety and carbon-13 enrichment for theazacitosine moiety is encompassed herein.

In certain embodiments, one or more hydrogen(s) is/are enriched withdeuterium(s) and one or more carbon(s) is/are enriched with carbon-13,i.e., any combination of deuterium enrichment and carbon-13 enrichmentof 5-azacytidine is encompassed herein.

3. Synthesis of Isotopically Enriched Cytidine Analogs

The compounds described herein may be synthesized using any method knownto one of ordinary skill in the art. For example, particular compoundsdescribed herein are synthesized using standard synthetic organicchemistry techniques known to those of ordinary skill in the art. Insome embodiments, known procedures for the synthesis of 5-azacytidineare employed, wherein one or more of the reagents, starting materials,precursors, or intermediates are replaced by one or moreisotopically-enriched reagents, starting materials, precursors, orintermediates, including but not limited to one or moredeuterium-enriched reagents, starting materials, precursors, orintermediates, and/or one or more carbon-13-enriched reagents, startingmaterials, precursors, or intermediates. Isotopically enriched reagents,starting materials, precursors, or intermediates are commerciallyavailable or may be prepared by routine chemical reactions known to oneof skill in the art. In some embodiments, the routes are based on thosedisclosed in U.S. Pat. No. 7,038,038, which is incorporated herein byreference in its entirety.

In certain embodiments, a suitable isotopically enriched startingmaterial, such as a deuterium-enriched ribose, a deuterium-enriched5-azacytosine, a carbon-13-enriched ribose, and/or a carbon-13-enriched5-azacytosine, may be employed as the starting material in the followinggeneral scheme to prepare the corresponding deuterium and/or carbon-13enriched 5-azacytidine (See Scheme 1). Following the procedures in U.S.Pat. No. 7,038,038, 5-azacytosine is treated with hexamethyldisilazane(HMDS) to render a silylated 5-azacytosine. Tetraacetyl-D-ribose isprepared by reacting D-ribose with sodium acetate in acetic anhydride,following the procedures in Brown et al., Biochemical Preparations,1955, 4, 70-76. The silylated 5-azacytosine is coupled totetraacetyl-D-ribose in the presence of TMS-triflate, and the resultingprotected 5-azacytidine is treated with sodium methoxide in methanol toyield 5-azacytidine. See U.S. Pat. No. 7,038,038.

In some embodiments, one or more hydrogen positions in the riboseportion of 5-azacytidine are enriched with deuterium. Such 5-azacytidineanalogs may be prepared following Scheme 1 from a suitabledeuterium-enriched ribose, purchased from a commercial source orprepared following literature procedures. Specific examples ofdeuterium-enriched ribose starting material include, but are not limitedto, the following compounds listed in Table 1, which may be converted tothe corresponding deuterium-enriched 5-azacytidine analogs.

TABLE 1 Starting 5-Azacytidine Material Structure Source/ReferenceProduct D-Ribose-1-D

Cambridge Isotope Lab. I-1 D-Ribose-2-D

Cambridge Isotope Lab. I-2 D-Ribose-3-D

Omicron Biochemicals, Inc. I-3 D-Ribose-4-D

Omicron Biochemicals, Inc. I-4 D-Ribose-5,5′-D₂

Omicron Biochemicals, Inc. I-5 D-Ribose- 3,4,5,5′-D₄

Prepared following the procedures in J. Am. Chem. Soc. 1996, 118,7929-7940. I-6

In other embodiments, the hydrogen position on the 5-azacytosine ring of5-azacytidine is enriched with deuterium. Such 5-azacytidine analog maybe prepared, e.g., from deuterated 5-azacytosine following Scheme 1. Thedeuterated 5-azacytosine may be prepared, e.g., from suitable deuteratedreagents as shown in Scheme 2. See e.g., Grundmann et al., Chem. Ber.1954, 87, 19-24; Piskala et al., in Zorbach and Tipson (eds.) SyntheticProcedures in Nucleic Acid Chemistry, Vol. 1, Wiley Interscience, NewYork, 1968, 107-108; Piskala, Collect. Czech. Chem. Comm. 1967, 32,3966-3976.

In other embodiments, both the hydrogen position on the 5-azacytosinering and one or more hydrogen positions in the ribose portion of5-azacytidine are enriched with deuterium. Such 5-azacytidine analogsmay be prepared, e.g., following Scheme 1, coupling a suitabledeuterated ribose starting materials with deuterated 5-azacytosine. Forexample, compounds I-9, I-10, I-11, I-12, I-13, and I-14 may be preparedfrom the corresponding deuterated ribose starting material listed inTable 1, and deuterated 5-azacytosine prepared according to Scheme 2.

In some embodiments, one or more carbon atoms in the ribose portion of5-azacytidine are enriched with carbon-13. Such 5-azacytidine analogsmay be prepared following Scheme 1 from a suitable carbon-13-enrichedribose, purchased from a commercial source or prepared followingliterature procedures. Specific examples of carbon-13-enriched ribosestarting material include, but are not limited to, the followingcompounds listed in Table 2, which may be converted to the correspondingcarbon-13-enriched 5-azacytidine analogs. (The asterisk “*” indicates acarbon-13 enriched atomic position)

TABLE 2 Starting 5-Azacytidine Material Structure Source/ReferenceProduct D-Ribose-1-¹³C

Sigma Aldrich II-1 D-Ribose-2-¹³C

Sigma Aldrich II-2 D-Ribose-3-¹³C

Omicron Biochemicals, Inc. II-3 D-Ribose-4-¹³C

Omicron Biochemicals, Inc. II-4 D-Ribose-5-¹³C

Cambridge Isotope Lab. II-5 D-Ribose-1,2-¹³C₂

Sigma Aldrich II-6 D-Ribose-1,3-¹³C₂

Omicron Biochemicals, Inc. II-7 D-Ribose-1,5-¹³C₂

Omicron Biochemicals, Inc. II-8 D-Ribose-2,5-¹³C₂

Omicron Biochemicals, Inc. II-9 D-Ribose- 2,3,4,5-¹³C₄

Sigma Aldrich  II-10 D-Ribose- 1,2,3,4,5-¹³C₅

Cambridge Isotope Lab.  II-11

In other embodiments, one or more carbon atoms in the 5-azacytosine ringare enriched with carbon-13. Such 5-azacytidine analogs may be preparedfrom a carbon-13-enriched 5-azacytosine following Scheme 1. Thecarbon-13 enriched 5-azacytosine intermediates may be prepared fromsuitable carbon-13 enriched reagents as shown in Scheme 3. See e.g.,Grundmann et al., Chem. Ber. 1954, 87, 19-24; Piskala et al., in Zorbachand Tipson (eds.) Synthetic Procedures in Nucleic Acid Chemistry, Vol.1, Wiley Interscience, New York, 1968, 107-108; Piskala, Collect. Czech.Chem. Comm. 1967, 32, 3966-3976.

In other embodiments, one or more carbon positions on the 5-azacytosinering and one or more carbon positions in the ribose portion of5-azacytidine are enriched with carbon-13. Such 5-azacytidine analogsmay be prepared following Scheme 1, coupling a suitablecarbon-13-enriched ribose starting materials with a suitablecarbon-13-enriched 5-azacytosine. For example, compounds may be preparedfrom a carbon-13-enriched ribose starting material listed in Table 2,and carbon-13-enriched 5-azacytosine prepared according to Scheme 3.

The routes and methods described above may be modified to provide anisotopolougue of 5-azacytidine having both deuterium enrichment andcarbon-13 enrichment.

C. Pharmaceutical Formulations

1. Overview

Embodiments herein encompass pharmaceutical formulations andcompositions comprising one or more cytidine analogs, e.g.,5-azacytidine, and optionally a permeation enhancer, wherein theformulations and compositions are prepared for oral administration. In aparticular embodiment, the formulations and compositions are preparedfor release of the cytidine analog substantially in the stomach. Inspecific embodiments, the cytidine analogs, e.g., 5-azacytidine, and thepharmaceutical formulations and compositions are used for treatingdiseases and disorders associated with abnormal cell proliferation,wherein the cytidine analogs, the formulations and compositions areprepared for oral administration, preferably for release of the cytidineanalogs substantially in the stomach. Particular embodiments relate tothe use of one or more cytidine analogs, e.g., 5-azacytidine, for thepreparation of pharmaceutical formulations and compositions for treatingparticular medical indications, as provided herein. The pharmaceuticalformulations and compositions comprising cytidine analogs providedherein are intended for oral delivery of the cytidine analog in subjectsin need thereof. Oral delivery formats include, but are not limited to,tablets, capsules, caplets, solutions, suspensions, and syrups, and mayalso comprise a plurality of granules, beads, powders or pellets thatmay or may not be encapsulated. Such formats may also be referred toherein as the “drug core” which contains the cytidine analog.

Particular embodiments herein provide solid oral dosage forms that aretablets or capsules. In certain embodiments, the formulation is a tabletcomprising a cytidine analog. In certain embodiments, the formulation isa capsule comprising a cytidine analog. In certain embodiments, thetablets or capsules provided herein optionally comprise one or moreexcipients, such as, for example, glidants, diluents, lubricants,colorants, disintegrants, granulating agents, binding agents, polymers,and coating agents. In certain embodiments, the formulation is animmediate release tablet. In certain embodiments, the formulation is acontrolled release tablet releasing the API, e.g., substantially in thestomach. In certain embodiments, the formulation is a hard gelatincapsule. In certain embodiments, the formulation is a soft gelatincapsule. In certain embodiments, the capsule is a hydroxypropylmethylcellulose (HPMC) capsule. In certain embodiments, the formulationis an immediate release capsule. In certain embodiments, the formulationis an immediate or controlled release capsule releasing the API, e.g.,substantially in the stomach. In certain embodiments, the formulation isa rapidly disintegrating tablet that dissolves substantially in themouth following administration. In certain embodiments, embodimentsherein encompass the use of cytidine analogs, e.g., 5-azacytidine, forthe preparation of a pharmaceutical composition for treating a diseaseassociated with abnormal cell proliferation, wherein the composition isprepared for oral administration.

2. Performance of Certain Dosage Forms Provided Herein

In certain embodiments, the formulations comprising the cytidineanalogs, such as, for example, 5-azacytidine, effect an immediaterelease of the API upon oral administration. In particular embodiments,the formulations comprising the cytidine analogs, such as, for example,5-azacytidine, comprise a therapeutically or prophylactically effectiveamount of the cytidine analog (and, optionally, one or more excipients)and effect an immediate release of the API upon oral administration.

In certain embodiments, the formulations comprising the cytidineanalogs, such as, for example, 5-azacytidine, effect a controlledrelease of the API substantially in the stomach upon oraladministration. In certain embodiments, the formulations comprising thecytidine analogs, such as, for example, 5-azacytidine, comprise atherapeutically or prophylactically effective amount of the cytidineanalog and a drug release controlling component which is capable ofreleasing the cytidine analog substantially in the stomach. In certainembodiments, matrices (e.g., polymer matrices) may be employed in theformulation to control the release of the cytidine analog. In certainembodiments, coatings and/or shells may be employed in the formulationto control the release of the cytidine analog in the substantially inthe stomach.

In certain embodiments, the formulations comprising the cytidineanalogs, such as, for example, 5-azacytidine, release the APIsubstantially in the stomach upon oral administration. In certainembodiments, the formulations effect an immediate release of thecytidine analog upon oral administration. In certain embodiments, theformulations optionally further comprises a drug release controllingcomponent, wherein the drug release controlling component is adjustedsuch that the release of the cytidine analog occurs substantially in thestomach. In particular embodiments, the drug release controllingcomponent is adjusted such that the release of the cytidine analog isimmediate and occurs substantially in the stomach. In particularembodiments, the drug release controlling component is adjusted suchthat the release of the cytidine analog is sustained and occurssubstantially in the stomach. In certain embodiments, the formulation ofthe cytidine analog, such as, for example, 5-azacytidine, releases theAPI substantially in the stomach, and, subsequently, releases theremainder of the API in the intestine upon oral administration.

Methods by which skilled practitioners can assess where a drug isreleased in the gastrointestinal tract of a subject are known in theart, and include, for example, scintigraphic studies, testing in abio-relevant medium which simulates the fluid in relevant portions ofthe gastrointestinal tract, among other methods.

Particular embodiments herein provide pharmaceutical formulations (e.g.,immediate release oral formulations and/or formulations that release theAPI substantially in the stomach) comprising a cytidine analog (e.g.,5-azacytidine) that achieve a particular exposure in the subject towhich the formulation is orally administered, as compared to a SC doseof the same cytidine analog. Particular embodiments provide oralformulations that achieve an exposure of at least about 5%, at leastabout 10%, at least about 15%, at least about 20%, at least about 25%,at least about 30%, at least about 35%, at least about 40%, at leastabout 45%, at least about 50%, at least about 55%, at least about 60%,at least about 65%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,or about 100%, as compared to a SC dose.

In certain embodiments, the formulation (e.g., immediate release oralformulation and/or formulation that release the API substantially in thestomach) comprising the cytidine analog, such as, for example,5-azacytidine, renders a certain percentage of the cytidine analog inthe formulation systemically bioavailable upon oral administration. Incertain embodiments, after the subject is orally administered theformulation, the cytidine analog in the formulation is absorbedsubstantially in the stomach, and becomes available to the body throughsystemic exposure. In particular embodiments, the oral bioavailabilityof a formulation comprising a cytidine analog provided herein is, e.g.,greater than about 1%, greater than about 5%, greater than about 10%,greater than about 15%, greater than about 20%, greater than about 25%,greater than about 30%, greater than about 35%, greater than about 40%,greater than about 45%, greater than about 50%, greater than about 55%,greater than about 60%, greater than about 65%, greater than about 70%,greater than about 75%, greater than about 80%, greater than about 85%,greater than about 90%, greater than about 95%, or about 100%, of thetotal amount of the cytidine analog in the formulation.

Methods by which skilled practitioners can assess the oralbioavailability of a drug formulation in a subject are known in the art.Such methods, include, for example, comparing certain dosing-relatedparameters, such as, but not limited to, maximum plasma concentration(“Cmax”), time to maximum plasma concentration (“Tmax”), orarea-under-the-curve (“AUC”) determinations.

Particular embodiments herein provide pharmaceutical formulations (e.g.,immediate release oral formulations and/or formulations that release theAPI substantially in the stomach) comprising a cytidine analog (e.g.,5-azacytidine) that achieve a particular AUC value (e.g., AUC(0-t) orAUC(0-∞)) in the subject (e.g., human) to which the formulation isorally administered. Particular embodiments provide oral formulationsthat achieve an AUC value of at least about 25 ng-hr/mL, at least about50 ng-hr/mL, at least about 75 ng-hr/mL, at least about 100 ng-hr/mL, atleast about 150 ng-hr/mL, at least about 200 ng-hr/mL, at least about250 ng-hr/mL, at least about 300 ng-hr/mL, at least about 350 ng-hr/mL,at least about 400 ng-hr/mL, at least about 450 ng-hr/mL, at least about500 ng-hr/mL, at least about 550 ng-hr/mL, at least about 600 ng-hr/mL,at least about 650 ng-hr/mL, at least about 700 ng-hr/mL, at least about750 ng-hr/mL, at least about 800 ng-hr/mL, at least about 850 ng-hr/mL,at least about 900 ng-hr/mL, at least about 950 ng-hr/mL, at least about1000 ng-hr/mL, at least about 1100 ng-hr/mL, at least about 1200ng-hr/mL, at least about 1300 ng-hr/mL, at least about 1400 ng-hr/mL, atleast about 1500 ng-hr/mL, at least about 1600 ng-hr/mL, at least about1700 ng-hr/mL, at least about 1800 ng-hr/mL, at least about 1900ng-hr/mL, at least about 2000 ng-hr/mL, at least about 2250 ng-hr/mL, orat least about 2500 ng-hr/mL. In particular embodiments, the AUCdetermination is obtained from a time-concentration pharmacokineticprofile obtained from the blood samples of animals or human volunteersfollowing dosing.

Particular embodiments herein provide pharmaceutical formulations (e.g.,immediate release oral formulations and/or formulations that release theAPI substantially in the stomach) comprising a cytidine analog (e.g.,5-azacytidine) that achieve a particular maximum plasma concentration(“Cmax”) in the subject to which the formulation is orally administered.Particular embodiments provide oral formulations that achieve a Cmax ofthe cytidine analog of at least about 25 ng/mL, at least about 50 ng/mL,at least about 75 ng/mL, at least about 100 ng/mL, at least about 150ng/mL, at least about 200 ng/mL, at least about 250 ng/mL, at leastabout 300 ng/mL, at least about 350 ng/mL, at least about 400 ng/mL, atleast about 450 ng/mL, at least about 500 ng/mL, at least about 550ng/mL, at least about 600 ng/mL, at least about 650 ng/mL, at leastabout 700 ng/mL, at least about 750 ng/mL, at least about 800 ng/mL, atleast about 850 ng/mL, at least about 900 ng/mL, at least about 950ng/mL, at least about 1000 ng/mL, at least about 1100 ng/mL, at leastabout 1200 ng/mL, at least about 1300 ng/mL, at least about 1400 ng/mL,at least about 1500 ng/mL, at least about 1600 ng/mL, at least about1700 ng/mL, at least about 1800 ng/mL, at least about 1900 ng/mL, atleast about 2000 ng/mL, at least about 2250 ng/mL, or at least about2500 ng/mL.

Particular embodiments herein provide pharmaceutical formulations (e.g.,immediate release oral formulations and/or formulations that release theAPI substantially in the stomach) comprising a cytidine analog (e.g.,5-azacytidine) that achieve a particular time to maximum plasmaconcentration (“Tmax”) in the subject to which the formulation is orallyadministered. Particular embodiments provide oral formulations thatachieve a Tmax of the cytidine analog of less than about 10 min., lessthan about 15 min., less than about 20 min., less than about 25 min.,less than about 30 min., less than about 35 min., less than about 40min., less than about 45 min., less than about 50 min., less than about55 min., less than about 60 min., less than about 65 min., less thanabout 70 min., less than about 75 min., less than about 80 min., lessthan about 85 min., less than about 90 min., less than about 95 min.,less than about 100 min., less than about 105 min., less than about 110min., less than about 115 min., less than about 120 min., less thanabout 130 min., less than about 140 min., less than about 150 min., lessthan about 160 min., less than about 170 min., less than about 180 min.,less than about 190 min., less than about 200 min., less than about 210min., less than about 220 min., less than about 230 min., or less thanabout 240 min. In particular embodiments, the Tmax value is measuredfrom the time at which the formulation is orally administered.

Particular embodiments herein provide oral dosage forms comprising acytidine analog, wherein the oral dosage forms have an enteric coating.Particular embodiments provide a permeable or partly permeable (e.g.,“leaky”) enteric coating with pores. In particular embodiments, thepermeable or partly permeable enteric-coated tablet releases the5-azacytidine in an immediate release manner substantially in thestomach.

3. Design of Certain Dosage Forms Provided Herein

Provided herein are dosage forms designed to maximize the absorptionand/or efficacious delivery of certain cytidine analogs, e.g.,5-azacytidine, upon oral administration, e.g., for release substantiallyin the stomach. Accordingly, certain embodiments herein provide a solidoral dosage form of a cytidine analog, such as, for example,5-azacytidine, using pharmaceutical excipients designed for immediaterelease of the API upon oral administration, e.g., substantially in thestomach. Particular immediate release formulations comprise a specificamount of a cytidine analog and optionally one or more excipients. Incertain embodiments, the formulation may be an immediate release tabletor an immediate release capsule (such as, e.g., an HPMC capsule).

Provided herein are methods of making the formulations provided hereincomprising the cytidine analogs provided herein (e.g., immediate releaseoral formulations and/or formulations that release the API substantiallyin the stomach). In particular embodiments, the formulations providedherein may be prepared using conventional methods known to those skilledin the field of pharmaceutical formulation, as described, e.g., inpertinent textbooks. See, e.g., REMINGTON, THE SCIENCE AND PRACTICE OFPHARMACY, 20th Edition, Lippincott Williams & Wilkins, (2000); ANSEL etal., PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 7th Edition,Lippincott Williams & Wilkins, (1999); GIBSON, PHARMACEUTICALPREFORMULATION AND FORMULATION, CRC Press (2001).

In particular embodiments, formulations provided herein (e.g., immediaterelease oral formulations, formulations that release the APIsubstantially in the stomach, or rapidly disintegrating formulationsthat dissolve substantially in the mouth) comprise a cytidine analog,such as, for example, 5-azacytidine, in a specific amount. In particularembodiments, the specific amount of the cytidine analog in theformulation is, e.g., about 10 mg, about 20 mg, about 40 mg, about 60mg, about 80 mg, about 100 mg, about 120 mg, about 140 mg, about 160 mg,about 180 mg, about 200 mg, about 220 mg, least about 240 mg, about 260mg, about 280 mg, about 300 mg, about 320 mg, about 340 mg, about 360mg, about 380 mg, about 400 mg, about 420 mg, about 440 mg, about 460mg, about 480 mg, about 500 mg, about 600 mg, about 700 mg, about 800mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about1300 mg, about 1400 mg, about 1500 mg, about 1600 mg, about 1700 mg,about 1800 mg, about 1900 mg, about 2000 mg, about 2100 mg, about 2200mg, about 2300 mg, about 2400 mg, about 2500 mg, about 3000 mg, about4000 mg, or about 5000 mg. In particular embodiments, the specificamount of the cytidine analog in the formulation is, e.g., at leastabout 10 mg, at least about 20 mg, at least about 40 mg, at least about60 mg, at least about 80 mg, at least about 100 mg, at least about 120mg, at least about 140 mg, at least about 160 mg, at least about 180 mg,at least about 200 mg, at least about 220 mg, at least about 240 mg, atleast about 260 mg, at least about 280 mg, at least about 300 mg, atleast about 320 mg, at least about 340 mg, at least about 360 mg, atleast about 380 mg, at least about 400 mg, at least about 420 mg, atleast about 440 mg, at least about 460 mg, at least about 480 mg, atleast about 500 mg, at least about 600 mg, at least about 700 mg, atleast about 800 mg, at least about 900 mg, at least about 1000 mg, atleast about 1100 mg, at least about 1200 mg, at least about 1300 mg, atleast about 1400 mg, at least about 1500 mg, at least about 1600 mg, atleast about 1700 mg, at least about 1800 mg, at least about 1900 mg, atleast about 2000 mg, at least about 2100 mg, at least about 2200 mg, atleast about 2300 mg, at least about 2400 mg, at least about 2500 mg, atleast about 3000 mg, at least about 4000 mg, or at least about 5000 mg.

In certain embodiments, the formulation is a tablet, wherein the tabletis manufactured using standard, art-recognized tablet processingprocedures and equipment. In certain embodiments, the method for formingthe tablets is direct compression of a powdered, crystalline and/orgranular composition comprising the cytidine analog, alone or incombination with one or more excipients, such as, for example, carriers,additives, polymers, or the like. In certain embodiments, as analternative to direct compression, the tablets may be prepared using wetgranulation or dry granulation processes. In certain embodiments, thetablets are molded rather than compressed, starting with a moist orotherwise tractable material. In certain embodiments, compression andgranulation techniques are used.

In certain embodiments, the formulation is a capsule, wherein thecapsules may be manufactured using standard, art-recognized capsuleprocessing procedures and equipments. In certain embodiments, softgelatin capsules may be prepared in which the capsules contain a mixtureof the cytidine analog and vegetable oil or non-aqueous, water misciblematerials such as, for example, polyethylene glycol and the like. Incertain embodiments, hard gelatin capsules may be prepared containinggranules of the cytidine analog in combination with a solid pulverulentcarrier, such as, for example, lactose, saccharose, sorbitol, mannitol,potato starch, corn starch, amylopectin, cellulose derivatives, orgelatin. In certain embodiments, a hard gelatin capsule shell may beprepared from a capsule composition comprising gelatin and a smallamount of plasticizer such as glycerol. In certain embodiments, as analternative to gelatin, the capsule shell may be made of a carbohydratematerial. In certain embodiments, the capsule composition mayadditionally include polymers, colorings, flavorings and opacifiers asrequired. In certain embodiments, the capsule comprises HPMC.

In certain embodiments, the formulation of the cytidine analog, such as,for example, 5-azacytidine, is prepared using aqueous solvents withoutcausing significant hydrolytic degradation of the cytidine analog. Inparticular embodiments, the formulation of the cytidine analog, such as,for example, 5-azacytidine, is a tablet which contains a coating appliedto the drug core using aqueous solvents without causing significanthydrolytic degradation of the cytidine analog in the formulation. Incertain embodiments, water is employed as the solvent for coating thedrug core. In certain embodiments, the oral dosage form of the cytidineanalog is a tablet containing a film coat applied to the drug core usingaqueous solvents. In particular embodiments, water is employed as thesolvent for film-coating. In particular embodiments, the tabletcontaining the cytidine analog is film-coated using aqueous solventswithout effecting degradation of the pharmaceutical composition. Inparticular embodiments, water is used as the film coating solventwithout effecting degradation of the pharmaceutical composition. Incertain embodiments, an oral dosage form comprising 5-azacytidine and anaqueous film coating effects immediate drug release upon oral delivery.In certain embodiments, the oral dosage form comprising 5-azacytidineand an aqueous film coating effects controlled drug release to the uppergastrointestinal tract, e.g., the stomach, upon oral administration. Inparticular embodiments, a tablet with an aqueous-based film coatingcomprises 5-azacytidine as the API.

In certain embodiments, provided herein is a controlled releasepharmaceutical formulation for oral administration of a cytidine analogthat releases the cytidine analog substantially in the stomach,comprising: a) a specific amount of a cytidine analog; b) a drug releasecontrolling component for controlling the release of the cytidine analogsubstantially in the upper gastrointestinal tract, e.g., the stomach;and c) optionally one or more excipients. In certain embodiments, theoral dosage form comprising the cytidine analog is prepared as acontrolled release tablet or capsule which includes a drug corecomprising the pharmaceutical composition and optional excipients.Optionally, a “seal coat” or “shell” is applied. In certain embodiments,a formulation provided herein comprising a cytidine analog providedherein is a controlled release tablet or capsule, which comprises atherapeutically effective amount of the cytidine analog, a drug releasecontrolling component that controls the release of the cytidine analogsubstantially in the stomach upon oral administration, and optionally,one or more excipients.

Particular embodiments provide a drug release controlling component thatis a polymer matrix, which swells upon exposure to gastric fluid toeffect the gastric retention of the formulation and the sustainedrelease of the cytidine analog from the polymer matrix substantially inthe stomach. In certain embodiments, such formulations may be preparedby incorporating the cytidine analog into a suitable polymeric matrixduring formulation. Examples of such formulations are known in the art.See, e.g., Shell et al., U.S. Patent Publication No. 2002/0051820(application Ser. No. 09/990,061); Shell et al., U.S. Patent PublicationNo. 2003/0039688 (application Ser. No. 10/045,823); Gusler et al., U.S.Patent Publication No. 2003/0104053 (application Ser. No. 10/029,134),each of which is incorporated herein by reference in its entirety.

In certain embodiments, the drug release controlling component maycomprise a shell surrounding the drug-containing core, wherein the shellreleases the cytidine analog from the core by, e.g., permittingdiffusion of the cytidine analog from the core and promoting gastricretention of the formulation by swelling upon exposure to gastric fluidsto a size that is retained in the stomach. In certain embodiments, suchformulations may be prepared by first compressing a mixture of thecytidine analog and one or more excipients to form a drug core, andcompressing another powdered mixture over the drug core to form theshell, or enclosing the drug core with a capsule shell made of suitablematerials. Examples of such formulations are known in the art. See,e.g., Berner et al., U.S. Patent Publication No. 2003/0104062application Ser. No. 10/213,823), incorporated herein by reference inits entirety.

Certain embodiments herein provide oral dosage forms comprising acytidine analog, wherein the dosage form contains pores in theconventional enteric coating. In particular embodiments, the oral dosageform of the cytidine analog is a tablet that contains a permeable orpartly permeable (e.g., “leaky”) enteric coating with pores. Inparticular embodiments, the permeable or partly permeable enteric-coatedtablet controls the release of the cytidine analog from the tabletprimarily to the upper gastrointestinal tract, e.g., the stomach. Inparticular embodiments, the permeable or partly permeable enteric-coatedtablet comprises 5-azacytidine. In particular embodiments, the remainderof the cytidine analog is subsequently released beyond the stomach(e.g., in the intestine).

In certain embodiments, the pharmaceutical formulation provided hereinis a compressed tablet comprising a cytidine analog. In addition to thecytidine analog, the tablet optionally comprises one or more excipients,including (a) diluents or fillers, which may add necessary bulk to aformulation to prepare tablets of the desired size; (b) binders oradhesives, which may promote adhesion of the particles of theformulation, enabling a granulation to be prepared and maintaining theintegrity of the final tablet; (c) disintegrants or disintegratingagents, which, after administration, may promote breakup of the tabletsto smaller particles for improved drug availability; (d) anti-adherents,glidants, lubricants or lubricating agents, which may enhance flow ofthe tableting material into the tablet dies, minimize wear of thepunches and dies, prevent the sticking of fill material to the punchesand dies, and produce tablets having a sheen; and (e) miscellaneousadjuncts such as colorants and flavorants. After compression, tabletsprovided herein may be coated with various materials as describedherein.

In certain embodiments, the pharmaceutical formulation provided hereinis a multiple compressed tablet of a cytidine analog. Multiplecompressed tablets are prepared by subjecting the fill material to morethan a single compression. The result may be a multiple-layered tabletor a tablet-within-a-tablet, the inner tablet being the core comprisinga cytidine analog and optionally one or more excipients, and the outerportion being the shell, wherein the shell comprises one or moreexcipients, and may or may not contain the cytidine analog. Layeredtablets may be prepared by the initial compaction of a portion of fillmaterial in a die followed by additional fill material and compressionto form two- or three-layered tablets, depending upon the number ofseparate fills. Each layer may contain a different therapeutic agent,separate from one another for reasons of chemical or physicalincompatibility, or the same therapeutic agent for staged drug release,or simply for the unique appearance of the multiple-layered tablet. Eachportion of fill may be colored differently to prepare a distinctivelooking tablet. In the preparation of tablets having a compressed tabletas the inner core, special machines may be used to place the preformedtablet precisely within the die for the subsequent compression ofsurrounding fill material.

In certain embodiments, the compressed tablet of a cytidine analog maybe coated with a colored or an uncolored sugar layer. The coating may bewater-soluble and quickly dissolved after oral ingestion. The sugarcoating may serve the purpose of protecting the enclosed drug from theenvironment and providing a barrier to an objectionable taste or smell.The sugar coating may also enhance the appearance of the compressedtablet and permit the imprinting of identifying manufacturer'sinformation. In certain embodiments, sugar-coated tablets may be 50%larger and heavier than the original uncoated tablets. The sugar-coatingof tablets may be divided into the following optional steps: (1)waterproofing and sealing (if needed); (2) sub-coating; (3) smoothingand final rounding; (4) finishing and coloring (if desired); (5)imprinting (if needed); and (6) polishing.

In certain embodiments, the compressed tablet of a cytidine analog maybe film-coated. Film-coated tablets may be compressed tablets coatedwith a thin layer of a polymer capable of forming a skin-like film overthe tablet. The film is usually colored and has the advantage to be moredurable, less bulky, and less time-consuming to apply. By itscomposition, the coating may be designed to rupture and expose the coretablet at the desired location within the gastrointestinal tract. Thefilm-coating process, which places a thin skin-tight coating of aplastic-like material over the compressed tablet, may produce coatedtablets having essentially the same weight, shape, and size as theoriginally compressed tablet. The film-coating may be colored to makethe tablets attractive and distinctive. Film-coating solutions may benon-aqueous or aqueous. In particular embodiments, the non-aqueoussolutions may optionally contain one or more of the following types ofmaterials to provide the desired coating to the tablets: (1) a filmformer capable of producing smooth, thin films reproducible underconventional coating conditions and applicable to a variety of tabletshapes, such as, for example, cellulose acetate phthalate; (2) analloying substance providing water solubility or permeability to thefilm to ensure penetration by body fluids and therapeutic availabilityof the drug, such as, for example, polyethylene glycol; (3) aplasticizer to produce flexibility and elasticity of the coating andthus provide durability, such as, for example, castor oil; (4) asurfactant to enhance spreadability of the film during application, suchas, for example, polyoxyethylene sorbitan derivatives; (5) opaquants andcolorants to make the appearance of the coated tablets attractive anddistinctive, such as, for example, titanium dioxide as an opaquant, andFD&C or D&C dyes as a colorant; (6) sweeteners, flavors, or aromas toenhance the acceptability of the tablet to the subject, such as, forexample, saccharin as sweeteners, and vanillin as flavors and aromas;(7) a glossant to provide a luster to the tablets without a separatepolishing operation, such as, for example, beeswax; and (8) a volatilesolvent to allow the spread of the other components over the tabletswhile allowing rapid evaporation to permit an effective yet speedyoperation, such as, for example, alcohol-acetone mixture. In certainembodiments, an aqueous film-coating formulation may contain one or moreof the following: (1) film-forming polymer, such as, for example,cellulose ether polymers as hydroxypropyl methyl-cellulose,hydroxypropyl cellulose, and methyl-cellulose; (2) plasticizer, such as,for example, glycerin, propylene glycol, polyethylene glycol, diethylphthalate, and dibutyl subacetate; (3) colorant and opacifier, such as,for example, FD&C or D&C lakes and iron oxide pigments; or (4) vehicle,such as, for example, water.

In certain embodiments, the compressed tablet of a cytidine analog maybe compression-coated. The coating material, in the form of agranulation or powder, may be compressed onto a tablet core of drug witha special tablet press.

In certain embodiments, the pharmaceutical formulation is agelatin-coated tablet of a cytidine analog. A gelatin-coated tablet is acapsule-shaped compressed tablet that allows the coated product to besmaller than a capsule filled with an equivalent amount of powder. Thegelatin coating facilitates swallowing and compared to unsealedcapsules, gelatin-coated tablets may be more tamper-evident.

In certain embodiments, the pharmaceutical formulation may be asublingual tablet of a cytidine analog. The sublingual tablet isintended to be dissolved beneath the tongue for absorption through theoral mucosa. The sublingual tablet may dissolve promptly and providerapid release of the drug.

In certain embodiments, the pharmaceutical formulation is an immediaterelease tablet of a cytidine analog. In certain embodiments, theimmediate release tablet is designed, e.g., to disintegrate and releasethe API absent of any special rate-controlling features, such as specialcoatings and other techniques. In certain embodiments, the formulationis a rapidly disintegrating tablet that, e.g., dissolves substantiallyin the mouth following administration. In certain embodiments, thepharmaceutical formulation is an extended release tablet of a cytidineanalog. In certain embodiments, the extended release tablet is designed,e.g., to release the API over an extended period of time andsubstantially in the stomach.

In certain embodiments, compressed tablets may be prepared by wetgranulation. Wet granulation is a widely employed method for theproduction of compressed tablets, and, in particular embodiments,requires one or more the following steps: (1) weighing and blending theingredients; (2) preparing a damp mass; (3) screening the damp mass intopellets or granules; (4) drying the granulation; (5) sizing thegranulation by dry screening; (6) adding lubricant and blending; and (7)tableting by compression.

In certain embodiments, compressed tablets may be prepared by drygranulation. By the dry granulation method, the powder mixture iscompacted in large pieces and subsequently broken down or sized intogranules. But this method, either the active ingredient or the diluenthas cohesive property. After weighing and mixing the ingredients, thepowder mixture may be slugged or compressed into large flat tablets orpellets. The slugs then are broken up by hand or by a mill and passedthrough a screen of desired mesh for sizing. Lubricant is added in theusual manner, and tablets are prepared by compression. Alternatively,instead of slugging, powder compactors may be used to increase thedensity of a powder by pressing it between high-pressure rollers. Thecompressed material then is broken up, sized, and lubricated, andtablets are prepared by compression in the usual manner. The rollercompaction method is often preferred over slugging. Binding agents usedin roller compaction formulations include methylcellulose orhydroxyl-methylcellulose and can produce good tablet hardness andfriability.

In certain embodiments, compressed tablets may be prepared by directcompression. Some granular chemicals possess free flowing and cohesiveproperties that enable them to be compressed directly in a tabletmachine without the need of wet or dry granulation. For chemicals thatdo not possess this quality, special pharmaceutical excipients may beused which impart the necessary qualities for the production of tabletsby direct compression. Particular tableting excipients include, e.g.:fillers, such as spray-dried lactose, micro-crystals ofalpha-monohydrate lactose, sucrose-invert sugar-corn starch mixtures,micro-crystalline cellulose, crystalline maltose, and di-calciumphosphate; disintegrating agents, such as direct-compression starch,sodium carboxymethyl starch, cross-linked carboxymethylcellulose fibers,and cross-linked polyvinylpyrrolidone; lubricants, such as magnesiumsearate and talc; and glidants, such as fumed silicon dioxide.

In certain embodiments, tablets provided herein may be prepared bymolding. The base for molded tablets is generally a mixture of finelypowdered lactose with or without a portion of powdered sucrose. Inpreparing the fill, the drug is mixed uniformly with the base bygeometric dilution. The powder mixture may be wetted with a mixture ofwater and alcohol sufficient only to dampen the powder so that it may becompacted. The solvent action of the water on a portion of thelactose/sucrose base effects the biding of the powder mixture upondrying. The alcohol portion hastens the drying process.

In certain embodiments, the pharmaceutical formulations provided hereincontain the cytidine analog and, optionally, one or more excipients toform a “drug core.” Optional excipients include, e.g., diluents (bulkingagents), lubricants, disintegrants, fillers, stabilizers, surfactants,preservatives, coloring agents, flavoring agents, binding agents,excipient supports, glidants, permeation enhancement excipients,plasticizers and the like, e.g., as known in the art. It will beunderstood by those in the art that some substances serve more than onepurpose in a pharmaceutical composition. For instance, some substancesare binders that help hold a tablet together after compression, yet arealso disintegrants that help break the tablet apart once it reaches thetarget delivery site. Selection of excipients and amounts to use may bereadily determined by the formulation scientist based upon experienceand consideration of standard procedures and reference works availablein the art.

In certain embodiments, formulations provided herein comprise one ormore binders. Binders may be used, e.g., to impart cohesive qualities toa tablet, and thus ensure that the tablet remains intact aftercompression. Suitable binders include, but are not limited to, starch(including corn starch and pregelatinized starch), gelatin, sugars(including sucrose, glucose, dextrose and lactose), polyethylene glycol,propylene glycol, waxes, and natural and synthetic gums, e.g., acaciasodium alginate, polyvinylpyrrolidone, cellulosic polymers (includinghydroxypropyl cellulose, hydroxypropylmethylcellulose, methyl cellulose,ethyl cellulose, hydroxyethyl cellulose, carboxymethyl cellulose and thelike), veegum, carbomer (e.g., carbopol), sodium, dextrin, guar gum,hydrogenated vegetable oil, magnesium aluminum silicate, maltodextrin,polymethacrylates, povidone (e.g., KOLLIDON, PLASDONE), microcrystallinecellulose, among others. Binding agents also include, e.g., acacia,agar, alginic acid, cabomers, carrageenan, cellulose acetate phthalate,ceratonia, chitosan, confectioner's sugar, copovidone, dextrates,dextrin, dextrose, ethylcellulose, gelatin, glyceryl behenate, guar gum,hydroxyethyl cellulose, hydroxyethylmethyl cellulose, hydroxypropylcellulose, hydroxypropyl starch, hypromellose, inulin, lactose,magnesium aluminum silicate, maltodextrin, maltose, methylcellulose,poloxamer, polycarbophil, polydextrose, polyethylene oxide,polymethylacrylates, povidone, sodium alginate, sodiumcarboxymethylcellulose, starch, pregelatinized starch, stearic acid,sucrose, and zein. The binding agent can be, relative to the drug core,in the amount of about 2% w/w of the drug core; about 4% w/w of the drugcore, about 6% w/w of the drug core, about 8% w/w of the drug core,about 10% w/w of the drug core, about 12% w/w of the drug core, about14% w/w of the drug core, about 16% w/w of the drug core, about 18% w/wof the drug core, about 20% w/w of the drug core, about 22% w/w of thedrug core, about 24% w/w of the drug core, about 26% w/w of the drugcore, about 28% w/w of the drug core, about 30% w/w of the drug core,about 32% w/w of the drug core, about 34% w/w of the drug core, about36% w/w of the drug core, about 38% w/w of the drug core, about 40% w/wof the drug core, about 42% w/w of the drug core, about 44% w/w of thedrug core, about 46% w/w of the drug core, about 48% w/w of the drugcore, about 50% w/w of the drug core, about 52% w/w of the drug core,about 54% w/w of the drug core, about 56% w/w of the drug core, about58% w/w of the drug core, about 60% w/w of the drug core, about 62% w/wof the drug core, about 64% w/w of the drug core, about 66% w/w of thedrug core; about 68% w/w of the drug core, about 70% w/w of the drugcore, about 72% w/w of the drug core, about 74% w/w of the drug core,about 76% w/w of the drug core, about 78% w/w of the drug core, about80% w/w of the drug core, about 82% w/w of the drug core, about 84% w/wof the drug core, about 86% w/w of the drug core, about 88% w/w of thedrug core, about 90% w/w of the drug core, about 92% w/w of the drugcore, about 94% w/w of the drug core, about 96% w/w of the drug core,about 98% w/w of the drug core, or more, if determined to beappropriate. In certain embodiments, a suitable amount of a particularbinder is determined by one of ordinary skill in the art.

In certain embodiments, formulations provided herein comprise one ormore diluents. Diluents may be used, e.g., to increase bulk so that apractical size tablet is ultimately provided. Suitable diluents includedicalcium phosphate, calcium sulfate, lactose, cellulose, kaolin,mannitol, sodium chloride, dry starch, microcrystalline cellulose (e.g.,AVICEL), microfine cellulose, pregelitinized starch, calcium carbonate,calcium sulfate, sugar, dextrates, dextrin, dextrose, dibasic calciumphosphate dihydrate, tribasic calcium phosphate, kaolin, magnesiumcarbonate, magnesium oxide, maltodextrin, mannitol, polymethacrylates(e.g., EUDRAGIT), potassium chloride, sodium chloride, sorbitol andtalc, among others. Diluents also include, e.g., ammonium alginate,calcium carbonate, calcium phosphate, calcium sulfate, celluloseacetate, compressible sugar, confectioner's sugar, dextrates, dextrin,dextrose, erythritol, ethylcellulose, fructose, fumaric acid, glycerylpalmitostearate, isomalt, kaolin, lacitol, lactose, mannitol, magnesiumcarbonate, magnesium oxide, maltodextrin, maltose, medium-chaintriglycerides, microcrystalline cellulose, microcrystalline silicifiedcellulose, powered cellulose, polydextrose, polymethylacrylates,simethicone, sodium alginate, sodium chloride, sorbitol, starch,pregelatinized starch, sucrose, sulfobutylether-β-cyclodextrin, talc,tragacanth, trehalose, and xylitol. Diluents may be used in amountscalculated to obtain a desired volume for a tablet or capsule; incertain embodiments, a diluent is used in an amount of about 5% or more,about 10% or more, about 15% or more, about 20% or more, about 22% ormore, about 24% or more, about 26% or more, about 28% or more, about 30%or more, about 32% or more, about 34% or more, about 36% or more, about38% or more, about 40% or more, about 42% or more, about 44% or more,about 46% or more, about 48% or more, about 50% or more, about 52% ormore, about 54% or more, about 56% or more, about 58% or more, about 60%or more, about 62% or more, about 64% or more, about 68% or more, about70% ore more, about 72% or more, about 74% or more, about 76% or more,about 78% or more, about 80% or more, about 85% or more, about 90% ormore, or about 95% or more, weight/weight, of a drug core; between about10% and about 90% w/w of the drug core; between about 20% and about 80%w/w of the drug core; between about 30% and about 70% w/w of the drugcore; between about 40% and about 60% w/w of the drug core. In certainembodiments, a suitable amount of a particular diluent is determined byone of ordinary skill in the art.

In certain embodiments, formulations provided herein comprise one ormore lubricants. Lubricants may be used, e.g., to facilitate tabletmanufacture; examples of suitable lubricants include, for example,vegetable oils such as peanut oil, cottonseed oil, sesame oil, oliveoil, corn oil, and oil of theobroma, glycerin, magnesium stearate,calcium stearate, and stearic acid. In certain embodiments, stearates,if present, represent no more than approximately 2 weight % of thedrug-containing core. Further examples of lubricants include, e.g.,calcium stearate, glycerin monostearate, glyceryl behenate, glycerylpalmitostearate, magnesium lauryl sulfate, magnesium stearate, myristicacid, palmitic acid, poloxamer, polyethylene glycol, potassium benzoate,sodium benzoate, sodium chloride, sodium lauryl sulfate, sodium stearylfumarate, stearic acid, talc, and zinc stearate. In particularembodiments, the lubricant is magnesium stearate. In certainembodiments, the lubricant is present, relative to the drug core, in anamount of about 0.2% w/w of the drug core, about 0.4% w/w of the drugcore, about 0.6% w/w of the drug core, about 0.8% w/w of the drug core,about 1.0% w/w of the drug core, about 1.2% w/w of the drug core, about1.4% w/w of the drug core, about 1.6% w/w of the drug core, about 1.8%w/w of the drug core, about 2.0% w/w of the drug core, about 2.2% w/w ofthe drug core, about 2.4% w/w of the drug core, about 2.6% w/w of thedrug core, about 2.8% w/w of the drug core, about 3.0% w/w of the drugcore, about 3.5% w/w of the drug core, about 4% w/w of the drug core,about 4.5% w/w of the drug core, about 5% w/w of the drug core, about 6%w/w of the drug core, about 7% w/w of the drug core, about 8% w/w of thedrug core, about 10% w/w of the drug core, about 12% w/w of the drugcore, about 14% w/w of the drug core, about 16% w/w of the drug core,about 18% w/w of the drug core, about 20% w/w of the drug core, about25% w/w of the drug core, about 30% w/w of the drug core, about 35% w/wof the drug core, about 40% w/w of the drug core, between about 0.2% andabout 10% w/w of the drug core, between about 0.5% and about 5% w/w ofthe drug core, or between about 1% and about 3% w/w of the drug core. Incertain embodiments, a suitable amount of a particular lubricant isdetermined by one of ordinary skill in the art.

In certain embodiments, formulations provided herein comprise one ormore disintegrants. Disintegrants may be used, e.g., to facilitatedisintegration of the tablet, and may be, e.g., starches, clays,celluloses, algins, gums or crosslinked polymers. Disintegrants alsoinclude, e.g., alginic acid, carboxymethylcellulose calcium,carboxymethylcellulose sodium (e.g., AC-DI-SOL, PRIMELLOSE), colloidalsilicon dioxide, croscarmellose sodium, crospovidone (e.g., KOLLIDON,POLYPLASDONE), guar gum, magnesium aluminum silicate, methyl cellulose,microcrystalline cellulose, polacrilin potassium, powdered cellulose,pregelatinized starch, sodium alginate, sodium starch glycolate (e.g.,EXPLOTAB) and starch. Additional disintegrants include, e.g., calciumalginate, chitosan, sodium docusate, hydroxypropyl cellulose, andpovidone. In certain embodiments, the disintegrant is, relative to thedrug core, present in the amount of about 1% w/w of the drug core, about2% w/w of the drug core, about 3% w/w of the drug core, about 4% w/w ofthe drug core, about 5% w/w of the drug core, about 6% w/w of the drugcore, about 7% w/w of the drug core, about 8% w/w of the drug core,about 9% w/w of the drug core, about 10% w/w of the drug core, about 12%w/w of the drug core, about 14% w/w of the drug core, about 16% w/w ofthe drug core, about 18% w/w of the drug core, about 20% w/w of the drugcore, about 22% w/w of the drug core, about 24% w/w of the drug core,about 26% w/w of the drug core, about 28% w/w of the drug core, about30% w/w of the drug core, about 32% w/w of the drug core, greater thanabout 32% w/w of the drug core, between about 1% and about 10% w/w ofthe drug core, between about 2% and about 8% w/w of the drug core,between about 3% and about 7% w/w of the drug core, or between about 4%and about 6% w/w of the drug core. In certain embodiments, a suitableamount of a particular disintegrant is determined by one of ordinaryskill in the art.

In certain embodiments, formulations provided herein comprise one ormore stabilizers. Stabilizers (also called absorption enhancers) may beused, e.g., to inhibit or retard drug decomposition reactions thatinclude, by way of example, oxidative reactions. Stabilizing agentsinclude, e.g., d-Alpha-tocopheryl polyethylene glycol 1000 succinate(Vitamin E TPGS), acacia, albumin, alginic acid, aluminum stearate,ammonium alginate, ascorbic acid, ascorbyl palmitate, bentonite,butylated hydroxytoluene, calcium alginate, calcium stearate, calciumcarboxymethylcellulose, carrageenan, ceratonia, colloidal silicondioxide, cyclodextrins, diethanolamine, edetates, ethylcellulose,ethyleneglycol palmitostearate, glycerin monostearate, guar gum,hydroxypropyl cellulose, hypromellose, invert sugar, lecithin, magnesiumaluminum silicate, monoethanolamine, pectin, poloxamer, polyvinylalcohol, potassium alginate, potassium polacrilin, povidone, propylgallate, propylene glycol, propylene glycol alginate, raffinose, sodiumacetate, sodium alginate, sodium borate, sodium carboxymethyl cellulose,sodium stearyl fumarate, sorbitol, stearyl alcohol,sufobutyl-b-cyclodextrin, trehalose, white wax, xanthan gum, xylitol,yellow wax, and zinc acetate. In certain embodiments, the stabilizer is,relative to the drug core, present in the amount of about 1% w/w of thedrug core, about 2% w/w of the drug core, about 3% w/w of the drug core,about 4% w/w of the drug core, about 5% w/w of the drug core, about 6%w/w of the drug core, about 7% w/w of the drug core, about 8% w/w of thedrug core, about 9% w/w of the drug core, about 10% w/w of the drugcore, about 12% w/w of the drug core, about 14% w/w of the drug core,about 16% w/w of the drug core, about 18% w/w of the drug core, about20% w/w of the drug core, about 22% w/w of the drug core, about 24% w/wof the drug core, about 26% w/w of the drug core, about 28% w/w of thedrug core, about 30% w/w of the drug core, about 32% w/w of the drugcore, between about 1% and about 10% w/w of the drug core, between about2% and about 8% w/w of the drug core, between about 3% and about 7% w/wof the drug core, or between about 4% and about 6% w/w of the drug core.In certain embodiments, a suitable amount of a particular stabilizer isdetermined by one of ordinary skill in the art.

In certain embodiments, formulations provided herein comprise one ormore glidants. Glidants may be used, e.g., to improve the flowproperties of a powder composition or granulate or to improve theaccuracy of dosing. Excipients that may function as glidants include,e.g., colloidal silicon dioxide, magnesium trisilicate, powderedcellulose, starch, tribasic calcium phosphate, calcium silicate,powdered cellulose, colloidal silicon dioxide, magnesium silicate,magnesium trisilicate, silicon dioxide, starch, tribasic calciumphosphate, and talc. In certain embodiments, the glidant is, relative tothe drug core, present in the amount of less than about 1% w/w of thedrug core, about 1% w/w of the drug core, about 2% w/w of the drug core,about 3% w/w of the drug core, about 4% w/w of the drug core, about 5%w/w of the drug core, about 6% w/w of the drug core, about 7% w/w of thedrug core, about 8% w/w of the drug core, about 9% w/w of the drug core,about 10% w/w of the drug core, about 12% w/w of the drug core, about14% w/w of the drug core, about 16% w/w of the drug core, about 18% w/wof the drug core, about 20% w/w of the drug core, about 22% w/w of thedrug core, about 24% w/w of the drug core, about 26% w/w of the drugcore, about 28% w/w of the drug core, about 30% w/w of the drug core,about 32% w/w of the drug core, between about 1% and about 10% w/w ofthe drug core, between about 2% and about 8% w/w of the drug core,between about 3% and about 7% w/w of the drug core, or between about 4%and about 6% w/w of the drug core. In certain embodiments, a suitableamount of a particular glidant is determined by one of ordinary skill inthe art.

In certain embodiments, formulations provided herein comprise one ormore permeation enhancers (also called, e.g., permeability enhancers).In certain embodiments, the permeation enhancer enhances the uptake of acytidine analog through the gastrointestinal wall (e.g., the stomach).In certain embodiments, the permeation enhancer alters the rate and/oramount of the cytidine analog that enters the bloodstream. In particularembodiments, d-alpha-tocopheryl polyethylene glycol-1000 succinate(Vitamin E TPGS) is used as a permeation enhancer. In particularembodiments, one or more other suitable permeation enhancers are used,including, e.g., any permeation enhancer known in the art. Specificexamples of suitable permeation enhancers include, e.g., those listedbelow:

Product name Chemical Name Example of Supplier Pluronic F 127 PoloxamerF 127 Sigma Lutrol F 68 Poloxamer 188 BASF Carbopol 934-P Carbomer 934-PSpectrum Chemical Tween 80 Polysorbate 80 Sigma Chitosan Chitosan LowMol Wt Aldrich Capric acid/Na cap Sodium Decanoate Sigma Lauric acid/Nalaur Sodium Dodecanoate Sigma Disodium EDTA Ethylenediamine tetraaceticSigma acid disodium dihydrate Propylene glycol 1, 2 Propanediol Sigma CMCellulose Carboxymethyl Cellulose Sigma Labrasol Caprylocaproylmacrogol- Gattefosse 8 glycerides N,N- (minimum 99%) SigmaDimethylacetamide Vitamin E TPGS d-Alpha-Tocopheryl Eastman PolyethyleneGlycol-1000 Succinate Solutol HS 15 Polyethylene glycol 660 BASF12-hydroxystearate Labrafil M Oleyl Macrogolglyerides Gattefosse 1944 CS(2)

Other potential permeation enhancers include, e.g., alcohols, dimethylsulfoxide, glyceryl monooleate, glycofurol, isopropyl myristate,isopropyl palmitate, lanolin, linoleic acid, myristic acid, oleic acid,oleyl alcohol, palmitic acid, polyoxyethylene alkyl ethers,2-pyrrolidone, sodium lauryl sulfate, and thymol.

In certain embodiments, the permeation enhancer is present in theformulation in an amount by weight, relative to the total weight of theformulation, of about 0.1%, about 0.2%, about 0.3%, about 0.4%, about0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about1.7%, about 1.8%, about 1.9%, about 2%, about 2.1%, about 2.2%, about2.3%, about 2.4%, about 2.5%, about 2.6%, about 2.7%, about 2.8%, about2.9%, about 3%, about 3.1%, about 3.2%, about 3.3%, about 3.4%, about3.5%, about 3.6%, about 3.7%, about 3.8%, about 3.9%, about 4%, about4.1% about 4.2%, about 4.3%, about 4.4%, about 4.5%, about 4.6%, about4.7%, about 4.8%, about 4.9%, about 5%, about 5.1% about 5.2%, about5.3%, about 5.4%, about 5.5%, about 5.6%, about 5.7%, about 5.8%, about5.9%, about 6%, about 6.1% about 6.2%, about 6.3%, about 6.4%, about6.5%, about 6.6%, about 6.7%, about 6.8%, about 6.9%, about 7%, about7.1% about 7.2%, about 7.3%, about 7.4%, about 7.5%, about 7.6%, about7.7%, about 7.8%, about 7.9%, about 8%, about 8.1% about 8.2%, about8.3%, about 8.4%, about 8.5%, about 8.6%, about 8.7%, about 8.8%, about8.9%, about 9%, about 9.1% about 9.2%, about 9.3%, about 9.4%, about9.5%, about 9.6%, about 9.7%, about 9.8%, about 9.9%, about 10%, greaterthan about 10%, greater than about 12%, greater than about 14%, greaterthan about 16%, greater than about 18%, greater than about 20%, greaterthan about 25%, greater than about 30%, greater than about 35%, greaterthan about 40%, greater than about 45%, or greater than about 50%. Incertain embodiments, the appropriate amount of a suitable permeationenhancer provided herein is determined by one of skill in the art.

Without intending to be limited to any particular theory, the permeationenhancers provided herein may function by, inter alia, facilitating(e.g., increasing the rate or extent of) the transport of a cytidineanalog through the gastrointestinal wall. In general, movement throughthe gastrointestinal wall may occur by, e.g.: passive diffusion, such asthe movement of drug across a membrane in a manner driven solely by theconcentration gradient; carrier-mediated diffusion, such as the movementof drug across a cell membrane via a specialized transport systemembedded in the cell membrane; paracellular diffusion, such as themovement of a drug across a membrane by going between, rather thanthrough, two cells; and transcellular diffusion, such as the movement ofa drug across the cell. Additionally, there are numerous cellularproteins capable of preventing intracellular accumulation of drugs bypumping out drug that enters the cell. These are sometimes called effluxpumps. One such efflux pump is that involving p-glycoprotein, which ispresent in many different tissues in the body (e.g., intestine,placental membrane, blood-brain barrier). Permeation enhancers canfunction by, inter alia, facilitating any of the processes mentionedabove (such as by increasing fluidity of membranes, opening tightjunctions between cells, and/or inhibiting efflux, among others).

In certain embodiments, the compositions provided herein comprising acytidine analog, e.g., 5-azacytidine, are essentially free of a cytidinedeaminase inhibitor (e.g., do not comprise a cytidine deaminaseinhibitor). In certain embodiments, the compositions provided herein areessentially free of (e.g., do not comprise) the cytidine deaminaseinhibitor tetrahydrouridine (THU). Certain embodiments herein providepharmaceutical compositions comprising a therapeutically effectiveamount of a cytidine analog (e.g., 5-azacytidine), wherein thecompositions release the cytidine analog substantially in the stomachfollowing oral administration to a subject, and wherein the compositionsare essentially free of (e.g., do not comprise) a cytidine deaminaseinhibitor (e.g., THU). Certain embodiments herein provide pharmaceuticalcompositions comprising a therapeutically effective amount of a cytidineanalog (e.g., 5-azacytidine), wherein the compositions release thecytidine analog substantially in the stomach following oraladministration to a subject, wherein the compositions are essentiallyfree of (e.g., do not comprise) a cytidine deaminase inhibitor (e.g.,THU), and wherein the compositions achieve a particular biologicalparameter provided herein (e.g., a particular Cmax value, Tmax value,and/or AUC value provided herein). In particular embodiments, acomposition provided herein that is essentially free of a cytidinedeaminase inhibitor (e.g., THU) comprises, e.g., less than 200 mg, lessthan 150 mg, less than 100 mg, less than 50 mg, less than 25 mg, lessthan 10 mg, less than 5 mg, less than 1 mg, or less than 0.1 mg of thecytidine deaminase inhibitor.

4. Additional Therapeutic Agents

In particular embodiments, the cytidine analog oral formulationsprovided herein further comprise one, two, three, or more otherpharmacologically active substances (also termed herein “additionaltherapeutic agents,” “second active agents,” or the like). In particularembodiments, the oral formulations provided herein comprise theadditional therapeutic agent(s) in a therapeutically effective amount.In particular embodiments, the cytidine analog (e.g., azacitidine) andthe additional therapeutic agent(s) are co-formulated together in thesame dosage form using methods of co-formulating active pharmaceuticalingredients, including methods disclosed herein and methods known in theart. In other embodiments, the cytidine analog and the additionaltherapeutic agent(s) are co-administered in separate dosage forms. It isbelieved that certain combinations work synergistically in the treatmentof particular diseases or disorders, including, e.g., types of cancerand certain diseases and conditions associated with, or characterizedby, undesired angiogenesis or abnormal cell proliferation. Cytidineanalog oral dosage forms provided herein can also work to alleviateadverse effects associated with certain second active agents, and somesecond active agents can be used to alleviate adverse effects associatedwith cytidine analog oral dosage forms provided herein. In certainembodiments, the oral formulations provided herein are co-administeredwith one or more therapeutic agents to provide a resensitization effectin subjects in need thereof. Additional therapeutic agents can be, e.g.,large molecules (e.g., proteins) or small molecules (e.g., syntheticinorganic, organometallic, or organic molecules).

Examples of particular additional therapeutic agents useful in thecompositions and methods disclosed herein include, but are not limitedto, e.g., cytotoxic agents, anti-metabolites, antifolates, HDACinhibitors (e.g., entinostat, also known as SNDX-275 or MS-275; orvorinostat, also known as suberoylanilide hydroxamic acid (SAHA) orN-hydroxy-N′-phenyl-octanediamide), DNA intercalating agents, DNAcross-linking agents, DNA alkylating agents, DNA cleaving agents,topoisomerase inhibitors, CDK inhibitors, JAK inhibitors,anti-angiogenic agents, Bcr-Abl inhibitors, HER2 inhibitors, EGFRinhibitors, VEGFR inhibitors, PDGFR inhibitors, HGFR inhibitors, IGFRinhibitors, c-Kit inhibitors, Ras pathway inhibitors, PI3K inhibitors,multi-targeted kinase inhibitors, mTOR inhibitors, anti-estrogens,anti-androgens, aromatase inhibitors, somatostatin analogs, ERmodulators, anti-tubulin agents, vinca alkaloids, taxanes, HSPinhibitors, Smoothened antagonists, telomerase inhibitors, COX-2inhibitors, anti-metastatic agents, immunosuppressants, biologics suchas antibodies, and hormonal therapies. In particular embodiments, theco-administered therapeutic agent is an immunomodulatory compound, e.g.,thalidomide, lenalidomide, or pomalidomide. The co-administered agentmay be dosed, e.g., orally or by injection.

Other examples of additional therapeutic agents include, but are notlimited to, hematopoietic growth factor, a cytokine, an anti-canceragent, granulocyte colony-stimulating factor (G-CSF),granulocyte-macrophage colony-stimulating factor (GM-CSF),erythropoietin (EPO), interleukin (IL), interferon (IFN), oblimersen,melphalan, topotecan, pentoxifylline, taxotere, irinotecan,ciprofloxacin, doxorubicin, vincristine, dacarbazine, Ara-C,vinorelbine, prednisone, cyclophosphamide, bortezomib, arsenic trioxide.Such additional therapeutic agents are particularly useful in methodsand compositions disclosed herein including, but not limited to, thoserelating to treatment of multiple myeloma.

Other examples of additional therapeutic agents include, but are notlimited to, an antibody (e.g., rituximab, anti-CD33), hematopoieticgrowth factor, cytokine, anti-cancer agent, antibiotic, cox-2 inhibitor,immunomodulatory agent, immunosuppressive agent, corticosteroid, or apharmacologically active mutant or derivative thereof. See, e.g., S.Nand et al., Leukemia and Lymphoma, 2008, 49(11):2141-47 (describing aPhase II study involving the administration of a combination ofhydroxyurea, azacitidine and low dose gemtuzumab ozogamicin to elderlypatients with AML and high-risk MDS, and concluding that thiscombination appears to be a safe and effective regimen in the treatmentof AML and high risk MDS in this group of patients). Such additionaltherapeutic agents are particularly useful in methods and compositionsdisclosed herein including, but not limited to, those relating totreatment of the diseases and disorders disclosed herein.

Examples of large molecule active agents include, but are not limitedto, hematopoietic growth factors, cytokines, and monoclonal andpolyclonal antibodies. Typical large molecule active agents arebiological molecules, such as naturally occurring or artificially madeproteins. Proteins that are particularly useful include proteins thatstimulate the survival and/or proliferation of hematopoietic precursorcells and immunologically active poietic cells in vitro or in vivo.Others stimulate the division and differentiation of committed erythroidprogenitors in cells in vitro or in vivo. Particular proteins include,but are not limited to: interleukins, such as IL-2 (includingrecombinant IL-II (“rIL2”) and canarypox IL-2), IL-10, IL-12, and IL-18;interferons, such as interferon alfa-2a, interferon alfa-2b, interferonalfa-n1, interferon alfa-n3, interferon beta-I a, and interferon gamma-Ib; GM-CF and GM-CSF; and EPO.

Particular proteins that can be used in the methods and compositionsprovided herein include, but are not limited to: filgrastim, which issold in the United States under the trade name Neupogen® (Amgen,Thousand Oaks, Calif.); sargramostim, which is sold in the United Statesunder the trade name Leukine® (Immunex, Seattle, Wash.); and recombinantEPO, which is sold in the United States under the trade name Epogen®(Amgen, Thousand Oaks, Calif.).

Recombinant and mutated forms of GM-CSF can be prepared as described inU.S. Pat. Nos. 5,391,485; 5,393,870; and 5,229,496; all of which areincorporated herein by reference. Recombinant and mutated forms of G-CSFcan be prepared as described in U.S. Pat. Nos. 4,810,643; 4,999,291;5,528,823; and 5,580,755; all of which are incorporated herein byreference.

Embodiments herein encompass the use of native, naturally occurring, andrecombinant proteins. Particular embodiments encompass mutants andderivatives (e.g., modified forms) of naturally occurring proteins thatexhibit, in vivo, at least some of the pharmacological activity of theproteins upon which they are based. Examples of mutants include, but arenot limited to, proteins that have one or more amino acid residues thatdiffer from the corresponding residues in the naturally occurring formsof the proteins. Also encompassed by the term “mutants” are proteinsthat lack carbohydrate moieties normally present in their naturallyoccurring forms (e.g., nonglycosylated forms). Examples of derivativesinclude, but are not limited to, pegylated derivatives and fusionproteins, such as proteins formed by fusing IgG1 or IgG3 to the proteinor active portion of the protein of interest. See, e.g., Penichet, M. L.and Morrison, S. L., J. Immunol. Methods 248:91-101 (2001).

Antibodies that can be used in combination with oral formulationsdisclosed herein include monoclonal and polyclonal antibodies. Examplesof antibodies include, but are not limited to, trastuzumab (Herceptin®),rituximab (Rituxan®), bevacizumab (Avastin™), pertuzumab (Omnitarg™),tositumomab (Bexxar®), edrecolomab (Panorex®), and G250. Oralformulations disclosed herein can also comprise, be combined with, orused in combination with anti-TNF-α antibodies.

Large molecule active agents may be administered in the form ofanti-cancer vaccines. For example, vaccines that secrete, or cause thesecretion of, cytokines such as IL-2, G-CSF, and GM-CSF can be used inthe methods, pharmaceutical compositions, and kits provided herein. See,e.g., Emens, L. A., et al., Curr. Opinion Mol. Ther. 3(1):77-84 (2001).

In one embodiment, the additional therapeutic agent (e.g.,large-molecule compound or small-molecule compound) reduces, eliminates,or prevents an adverse effect associated with the administration (e.g.,oral administration) of a cytidine analog provided herein. Depending onthe particular cytidine analog and the disease or disorder begintreated, adverse effects can include, but are not limited to, anemia,neutropenia, febrile neutropenia, thrombocytopenia, hepatotoxicity(e.g., including, but not limited to, hepatoxicity in patients withpreexisting hepatic impairment), elevated serum creatinine, renalfailure, renal tubular acidosis, hypokalemia, hepatic coma, nausea,vomiting, dyspepsia, abdominal pain, pyrexia, leukopenia, diarrhea,constipation, ecchymosis, petechiae, rigors, weakness, pneumonia,anxiety, insomnia, lethargy, and decrease in weight, among others knownin the art to be associated with particular cytidine analogs.

Like some large molecules, many small-molecule compounds are believed tobe capable of providing a synergistic effect when administered with(e.g., before, after or simultaneously) a cytidine analog oralformulation disclosed herein. Examples of small molecule second activeagents include, but are not limited to, anti-cancer agents, antibiotics,immunosuppressive agents, and steroids.

Examples of anti-cancer agents include, but are not limited to:acivicin; aclarubicin; acodazole hydrochloride; acronine; adozelesin;aldesleukin; altretamine; ambomycin; ametantrone acetate; amsacrine;anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa;azotomycin; batimastat; benzodepa; bicalutamide; bisantrenehydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate;brequinar sodium; bropirimine; busulfan; cactinomycin; calusterone;caracemide; carbetimer; carboplatin; carmustine; carubicinhydrochloride; carzelesin; cedefingol; celecoxib (COX-2 inhibitor);chlorambucil; cirolemycin; cisplatin; cladribine; crisnatol mesylate;cyclophosphamide; cytarabine; dacarbazine; dactinomycin; daunorubicinhydrochloride; decitabine; dexormaplatin; dezaguanine; dezaguaninemesylate; diaziquone; docetaxel; doxorubicin; doxorubicin hydrochloride;droloxifene; droloxifene citrate; dromostanolone propionate; duazomycin;edatrexate; eflornithine hydrochloride; elsamitrucin; enloplatin;enpromate; epipropidine; epirubicin hydrochloride; erbulozole;esorubicin hydrochloride; estramustine; estramustine phosphate sodium;etanidazole; etoposide; etoposide phosphate; etoprine; fadrozolehydrochloride; fazarabine; fenretinide; floxuridine; fludarabinephosphate; fluorouracil; flurocitabine; fosquidone; fostriecin sodium;gemcitabine; gemcitabine hydrochloride; hydroxyurea; idarubicinhydrochloride; ifosfamide; ilmofosine; iproplatin; irinotecan;irinotecan hydrochloride; lanreotide acetate; letrozole; leuprolideacetate; liarozole hydrochloride; lometrexol sodium; lomustine;losoxantrone hydrochloride; masoprocol; maytansine; mechlorethaminehydrochloride; megestrol acetate; melengestrol acetate; melphalan;menogaril; mercaptopurine; methotrexate; methotrexate sodium; metoprine;meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin; mitomalcin;mitomycin; mitosper; mitotane; mitoxantrone hydrochloride; mycophenolicacid; nocodazole; nogalamycin; ormaplatin; oxisuran; paclitaxel;pegaspargase; peliomycin; pentamustine; peplomycin sulfate;perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride;plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine;procarbazine hydrochloride; puromycin; puromycin hydrochloride;pyrazofurin; riboprine; safingol; safingol hydrochloride; semustine;simtrazene; sparfosate sodium; sparsomycin; spirogermaniumhydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin;sulofenur; talisomycin; tecogalan sodium; taxotere; tegafur;teloxantrone hydrochloride; temoporfin; teniposide; teroxirone;testolactone; thiamiprine; thioguanine; thiotepa; tiazofurin;tirapazamine; toremifene citrate; trestolone acetate; triciribinephosphate; trimetrexate; trimetrexate glucuronate; triptorelin;tubulozole hydrochloride; uracil mustard; uredepa; vapreotide;verteporfin; vinblastine sulfate; vincristine sulfate; vindesine;vindesine sulfate; vinepidine sulfate; vinglycinate sulfate;vinleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate;vinzolidine sulfate; vorozole; zeniplatin; zinostatin; and zorubicinhydrochloride.

Other anti-cancer drugs include, but are not limited to: 20-epi-1,25dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclarubicin;acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TK antagonists;altretamine; ambamustine; amidox; amifostine; aminolevulinic acid;amrubicin; amsacrine; anagrelide; anastrozole; andrographolide;angiogenesis inhibitors; antagonist D; antagonist G; antarelix;anti-dorsalizing morphogenetic protein-1; antiandrogen, prostaticcarcinoma; antiestrogen; antineoplaston; antisense oligonucleotides;aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators;apurinic acid; ara-CDP-DL-PTBA; arginine deaminase; asulacrine;atamestane; atrimustine; axinastatin 1; axinastatin 2; axinastatin 3;azasetron; azatoxin; azatyrosine; baccatin III derivatives; balanol;batimastat; BCR/ABL antagonists; benzochlorins; benzoylstaurosporine;beta lactam derivatives; beta-alethine; betaclamycin B; betulinic acid;bFGF inhibitor; bicalutamide; bisantrene; bisaziridinylspermine;bisnafide; bistratene A; bizelesin; breflate; bropirimine; budotitane;buthionine sulfoximine; calcipotriol; calphostin C; camptothecinderivatives; capecitabine; carboxamide-amino-triazole;carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor;carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropinB; cetrorelix; chlorlns; chloroquinoxaline sulfonamide; cicaprost;cis-porphyrin; cladribine; clomifene analogues; clotrimazole;collismycin A; collismycin B; combretastatin A4; combretastatinanalogue; conagenin; crambescidin 816; crisnatol; cryptophycin 8;cryptophycin A derivatives; curacin A; cyclopentanthraquinones;cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor;cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin;dexamethasone; dexifosfamide; dexrazoxane; dexverapamil; diaziquone;didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine;dihydrotaxol, 9-; dioxamycin; diphenyl spiromustine; docetaxel;docosanol; dolasetron; doxifluridine; doxorubicin; droloxifene;dronabinol; duocarmycin SA; ebselen; ecomustine; edelfosine;edrecolomab; eflornithine; elemene; emitefur; epirubicin; epristeride;estramustine analogue; estrogen agonists; estrogen antagonists;etanidazole; etoposide phosphate; exemestane; fadrozole; fazarabine;fenretinide; filgrastim; finasteride; flavopiridol; flezelastine;fluasterone; fludarabine; fluorodaunorunicin hydrochloride; forfenimex;formestane; fostriecin; fotemustine; gadolinium texaphyrin; galliumnitrate; galocitabine; ganirelix; gelatinase inhibitors; gemcitabine;glutathione inhibitors; hepsulfam; heregulin; hexamethylenebisacetamide; hypericin; ibandronic acid; idarubicin; idoxifene;idramantone; ilmofosine; ilomastat; imatinib (e.g., Gleevec®),imiquimod; immunostimulant peptides; insulin-like growth factor-1receptor inhibitor; interferon agonists; interferons; interleukins;iobenguane; iododoxorubicin; ipomeanol, 4-; iroplact; irsogladine;isobengazole; isohomohalicondrin B; itasetron; jasplakinolide;kahalalide F; lamellarin-N triacetate; lanreotide; leinamycin;lenograstim; lentinan sulfate; leptolstatin; letrozole; leukemiainhibiting factor; leukocyte alpha interferon;leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole;linear polyamine analogue; lipophilic disaccharide peptide; lipophilicplatinum compounds; lissoclinamide 7; lobaplatin; lombricine;lometrexol; lonidamine; losoxantrone; loxoribine; lurtotecan; lutetiumtexaphyrin; lysofylline; lytic peptides; maitansine; mannostatin A;marimastat; masoprocol; maspin; matrilysin inhibitors; matrixmetalloproteinase inhibitors; menogaril; merbarone; meterelin;methioninase; metoclopramide; MIF inhibitor; mifepristone; miltefosine;mirimostim; mitoguazone; mitolactol; mitomycin analogues; mitonafide;mitotoxin fibroblast growth factor-saporin; mitoxantrone; mofarotene;molgramostim; Erbitux, human chorionic gonadotrophin; monophosphoryllipid A+myobacterium cell wall sk; mopidamol; mustard anticancer agent;mycaperoxide B; mycobacterial cell wall extract; myriaporone;N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip;naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin;nemorubicin; neridronic acid; nilutamide; nisamycin; nitric oxidemodulators; nitroxide antioxidant; nitrullyn; oblimersen (Genasense®);O⁶-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone;ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin;osaterone; oxaliplatin; oxaunomycin; paclitaxel; paclitaxel analogues;paclitaxel derivatives; palauamine; palmitoylrhizoxin; pamidronic acid;panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase;peldesine; pentosan polysulfate sodium; pentostatin; pentrozole;perflubron; perfosfamide; perillyl alcohol; phenazinomycin;phenylacetate; phosphatase inhibitors; picibanil; pilocarpinehydrochloride; pirarubicin; piritrexim; placetin A; placetin B;plasminogen activator inhibitor; platinum complex; platinum compounds;platinum-triamine complex; porfimer sodium; porfiromycin; prednisone;propyl bis-acridone; prostaglandin J2; proteasome inhibitors; proteinA-based immune modulator; protein kinase C inhibitor; protein kinase Cinhibitors, microalgal; protein tyrosine phosphatase inhibitors; purinenucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine;pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists;raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors;ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re186 etidronate; rhizoxin; ribozymes; RII retinamide; rohitukine;romurtide; roquinimex; rubiginone B1; ruboxyl; safingol; saintopin;SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics; semustine;senescence derived inhibitor 1; sense oligonucleotides; signaltransduction inhibitors; sizofiran; sobuzoxane; sodium borocaptate;sodium phenylacetate; solverol; somatomedin binding protein; sonermin;sparfosic acid; spicamycin D; spiromustine; splenopentin; spongistatin1; squalamine; stipiamide; stromelysin inhibitors; sulfinosine;superactive vasoactive intestinal peptide antagonist; suradista;suramin; swainsonine; tallimustine; tamoxifen methiodide; tauromustine;tazarotene; tecogalan sodium; tegafur; tellurapyrylium; telomeraseinhibitors; temoporfin; teniposide; tetrachlorodecaoxide; tetrazomine;thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic;thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroidstimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocenebichloride; topsentin; toremifene; translation inhibitors; tretinoin;triacetyluridine; triciribine; trimetrexate; triptorelin; tropisetron;turosteride; tyrosine kinase inhibitors; tyrphostins; UBC inhibitors;ubenimex; urogenital sinus-derived growth inhibitory factor; urokinasereceptor antagonists; vapreotide; variolin B; velaresol; veramine;verdins; verteporfin; vinorelbine; vinxaltine; vitaxin; vorozole;zanoterone; zeniplatin; zilascorb; and zinostatin stimalamer.

Specific additional therapeutic agents include, but are not limited to,oblimersen (Genasense®), remicade, docetaxel, celecoxib, melphalan,dexamethasone (Decadron®), steroids, gemcitabine, cisplatinum,temozolomide, etoposide, cyclophosphamide, temodar, carboplatin,procarbazine, gliadel, tamoxifen, topotecan, methotrexate, Arisa®,taxol, taxotere, fluorouracil, leucovorin, irinotecan, xeloda, CPT-11,interferon alpha, pegylated interferon alpha (e.g., PEG INTRON-A),capecitabine, cisplatin, thiotepa, fludarabine, carboplatin, liposomaldaunorubicin, cytarabine, doxetaxol, pacilitaxel, vinblastine, IL-2,GM-CSF, dacarbazine, vinorelbine, zoledronic acid, palmitronate, biaxin,busulphan, prednisone, bisphosphonate, arsenic trioxide, vincristine,doxorubicin (Doxil®), paclitaxel, ganciclovir, adriamycin, estramustinesodium phosphate (Emcyt®), sulindac, and etoposide.

Specific additional therapeutic agents include R-CHOP, which is a courseof rituximab, doxorubicin, cyclophosphamide, vincristine, andprednisone. Chemotherapy is given as a course of several cycles oftreatment over a few months. Each cycle of R-CHOP is 21 days (threeweeks). On the first day, rituximab, doxorubicin, vincristine andcyclophosphamide are given, and a five-day course of prednisone tabletsis started at the end of the 21 days. A second cycle of R-CHOP isstarted, and up to eight cycles over 3-4 months may be given asdetermined by the medical professional.

In one embodiment, rituximab, doxorubicin, vincristine andcyclophosphamide are administered on the first day of the 21 day cycle,and a five-day course of prednisone tablets is also started on Days 1-5of the 21 day cycle. A second cycle of R-CHOP is started, and up toeight cycles over 3-4 months may be given as determined by the medicalprofessional.

D. Methods of Use

As described herein, certain embodiments herein provide oralformulations of cytidine analogs useful in methods relating to, e.g.,permitting different dosing amounts and/or dosing periods; providingalternative pharmacokinetic profiles, pharmacodynamic profiles, and/orsafety profiles; permitting the evaluation of long-term and/ormaintenance therapies; providing treatment regimens that maximizedemethylation and/or gene re-expression; providing treatment regimensthat prolong continuous demethylation; providing new indications forcytidine analogs; and/or providing other potential advantageousbenefits.

Provided herein are methods of treating patho-physiological conditionsmanifested by abnormal cell proliferation, such as, for example, cancer,including hematological disorders and solid tumors, by orallyadministering a pharmaceutical formulation comprising a cytidine analog,such as, for example, 5-azacytidine, wherein the formulation releasesthe cytidine analog substantially in the stomach. Other embodimentsherein provide methods of treating immune disorders. In particularembodiments, the methods provided herein involve oral administering aformulation that effects an immediate release of the cytidine analog. Incertain embodiments, the cytidine analog and one or more therapeuticagents are co-administered to subjects to yield a synergistictherapeutic effect. The co-administered agent may be a cancertherapeutic agent dosed orally or by injection.

In certain embodiments, methods provided herein for treating disordersrelated to abnormal cell proliferation comprise orally administering aformulation comprising a therapeutically effective amount of a cytidineanalog. Particular therapeutic indications relating to the methodsprovided herein are disclosed herein. In certain embodiments, thetherapeutically effective amount of the cytidine analog in thepharmaceutical formulation is an amount as disclosed herein. In certainembodiments, the precise therapeutically effective amount of thecytidine analog in the pharmaceutical formulation will vary dependingon, e.g., the age, weight, disease and/or condition of the subject.

In particular embodiments, the disorders related to abnormal cellproliferation include, but are not limited to, MDS, AML, ALL, CML,leukemia, chronic lymphocytic leukemia (CLL), lymphoma (includingnon-Hodgkin's lymphoma (NHL) and Hodgkin's lymphoma), multiple myeloma(MM), sarcoma, melanoma, carcinoma, adenocarcinoma, chordoma, breastcancer, colorectal cancer, ovarian cancer, lung cancer (e.g.,non-small-cell lung cancer and small-cell lung cancer), testicularcancer, renal cancer, pancreatic cancer, bone cancer, gastric cancer,head and neck cancer, and prostate cancer. In particular embodiment, thedisorder related to abnormal cell proliferation is MDS. In particularembodiments, the disorder related to abnormal cell proliferation is AML.

In certain embodiments, methods provided herein for treating disordersof abnormal cell proliferation comprise administering a cytidine analogusing at least two of IV, SC and oral administration methods. Forexample, particular embodiments herein provide administering an initialtreatment cycle of a cytidine analog, such as, for example,5-azacytidine, administered either SC or IV, followed by subsequentorally administered treatment cycles of the cytidine analog. In certainembodiments, treatment cycles comprise multiple doses administered to asubject in need thereof over multiple days (e.g., 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, or greater than 14 days), optionally followedby treatment dosing holidays (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, or greater than 14 days). Particular embodiments hereinprovide a treatment schedule comprising SC and/or IV administration forone, two, three, four, five, or more initial cycles, followed by oraladministration for subsequent cycles. For example, particularembodiments herein provide a treatment schedule comprising SCadministration for cycle 1, followed by oral administration forsubsequent cycles. Suitable dosage ranges and amounts for the methodsprovided herein are provided throughout the specification. For example,in certain embodiments, the SC dose is about 75 mg/m². In certainembodiments, the oral dose is about 60 mg, about 80 mg, about 120 mg,about 180 mg, about 240 mg, about 300 mg, about 360 mg, about 480 mg, orgreater than about 480 mg. In certain embodiments, oral doses arecalculated to achieve 80%, 100%, or 120% of SC AUC.

In certain embodiments, methods of treating disorders of abnormal cellproliferation comprises orally administering a formulation comprising acytidine analog (e.g., 5-azacytidine) as single or multiple daily doses.In particular embodiments, the formulation(s) comprising the cytidineanalog is/are orally administered once per day, twice per day, threetimes per day, four times per day, or more than four times per day. Forexample, in certain embodiments, the formulation comprising the cytidineanalog is administered using a treatment cycle comprising administrationof about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg,about 700 mg, about 800 mg, about 900 mg, or about 1,000 mg of thecytidine analog once, twice, three, or four times per day for 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, or 30 days. In certain embodiments, the method of treatingcomprises continuous low-dose administration. In certain embodiments,the formulation comprising the cytidine analog is administered using atreatment cycle comprising administration of about 300 mg of thecytidine analog twice per day for 7 days. In certain embodiments, theformulation comprising the cytidine analog is administered using atreatment cycle comprising administration of about 300 mg of thecytidine analog twice per day for 14 days. In certain embodiments, theformulation comprising the cytidine analog is administered using atreatment cycle comprising administration of about 300 mg of thecytidine analog three times per day for 7 days. In certain embodiments,the formulation comprising the cytidine analog is administered using atreatment cycle comprising administration of about 300 mg of thecytidine analog three times per day for 14 days. In certain embodiments,methods provided herein comprise administering a formulation comprisinga cytidine analog using one or more of the cycles provided herein, andrepeating one or more of the cycles for a period of, e.g., 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, or greater than 12 months.

In certain embodiments, methods herein comprise administering particularoral formulations provided herein to, e.g., overcome limitationsassociated with IV or SC administration of cytidine analogs. Forexample, IV or SC administration may limit the ability to deliver acytidine analog for longer periods of time on a regular basis, therebypotentially limiting the maximal efficacy of the cytidine analog. Due tothe difficulties of complying with the rigors of a prolonged IV or SCdosing schedule, prolonged SC or IV exposure to a cytidine analog maycause subjects (e.g., subjects with multiple cytopenias) to discontinuefrom the regimen. See, e.g., Lyons, R. M., et al., Hematologic Responseto Three Alternative Dosing Schedules of Azacitidine in Patients WithMyelodysplastic Syndromes, J. Clin. Oncol. (2009) (DOI:10.1200/JCO.2008.17.1058), which is incorporated by reference herein inits entirety. Accordingly, in certain embodiments, methods providedherein comprise administering an oral formulation provided herein toovercome these or other limitations associated with SC or IV cytidineanalog administration. For example, in certain embodiments, methodsprovided herein comprise administering daily to a subject an oralformulation provided herein for 7 or more, 8 or more, 9 or more, 10 ormore, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 ormore, 17 or more, 18 or more, 19 or more, 20 or more, or 21 or moredays.

Certain embodiments herein provide methods comprising administering oralformulations of cytidine analogs provided herein comprising deliveringthe cytidine analog (e.g., azacitidine) at a lower dose over a moreprolonged period of time, as compared to IV or SC administration. Inparticular embodiments, such methods comprise managing dose-relatedcytopenias (including, e.g., dose-related cytopenias associated withazacitidine) by administering an oral formulation provided herein. Incertain embodiments, methods provided herein comprise administering anoral formulation provided herein to achieve an improved safety profileas compared to an IV or SC dose comprising the same cytidine analog.

As described herein, certain embodiments provide methods for improvedtreatment of particular diseases or disorders (e.g., treatment of solidtumors) by administering an oral formulation provided herein, ascompared to IV or SC administration of the cytidine analog. Inparticular embodiments, certain methods herein provide administeringoral formulations provided herein at lower doses for more prolongedperiods of time, leading to improved demethylation. For example, certainmethods provided herein comprise administering an oral formulationprovided herein to treat a solid tumor while avoiding certaindose-limiting-toxicity-related side effects associated with dosing thecytidine analog via SC or IV administration. An example of certaintoxicity-related drawbacks associated with administration of a cytidineanalog are described, e.g., in K. Appleton et al., J. Clin. Oncol., Vol.25(29):4603-4609 (2007), which is incorporated by reference herein inits entirety.

Particular embodiments herein provide methods for treating a subjecthaving a disease or disorder provided herein by orally administering apharmaceutical composition provided herein, wherein the treatmentresults in improved survival of the subject. In certain embodiments, theimproved survival is measured as compared to one or more conventionalcare regimens. Particular embodiments herein provide methods fortreating a subject having a disease or disorder provided herein byorally administering a pharmaceutical composition provided herein,wherein the treatment provides improved effectiveness. In particularembodiments, the improved effectiveness is measured using one or moreendpoints for cancer clinical trials, as recommended by the U.S. Foodand Drug Administration (FDA). For example, FDA provides Guidance forIndustry on Clinical Trial Endpoints for the Approval of Cancer Drugsand Biologics (http://www.fda.gov/CbER/gdlns/clintrialend.htm). The FDAendpoints include, but are not limited to, Overall Survival, EndpointsBased on Tumor Assessments such as (i) Disease-Free Survival (ii)Objective Response Rate, (iii) Time to Progression and Progression-FreeSurvival and (iv) Time-to-Treatment Failure. Endpoints Involving SymptomEndpoints may include Specific Symptom Endpoints such as (i) Time toprogression of cancer symptoms and (ii) A composite symptom endpoint.Biomarkers assayed from blood or body fluids may also be useful todetermine the management of the disease.

In certain embodiments, the methods of treating disorders of abnormalcell proliferation comprise orally administering a formulation of acytidine analog with food. In certain embodiments, the methods oftreating disorders of abnormal cell proliferation comprise orallyadministering a formulation of a cytidine analog without food. Incertain embodiments, pharmacological parameters (e.g., Cmax, Tmax)depend on the fed state of the subject. In certain embodiments, theformulation of the cytidine analog is administered sublingually.

In certain embodiments, the cytidine analog, e.g., 5-azacytidine, is notco-administered with a cytidine deaminase inhibitor. In certainembodiments, the oral formulation comprising a cytidine analog asprovided herein is not co-administered with THU. Certain embodimentsherein provide methods of treating a disease or disorder provided herein(e.g., a disease associated with abnormal cell proliferation) comprisingorally administering a cytidine analog provided herein (e.g.,5-azacytidine) for release substantially in the stomach, wherein themethods achieve a particular biological parameter provided herein (e.g.,a particular Cmax value, Tmax value, and/or AUC value provided herein),and wherein the methods comprise not co-administering a cytidinedeaminase inhibitor with the cytidine analog. Certain embodiments hereinprovide methods of treating a disease or disorder provided herein (e.g.,a disease associated with abnormal cell proliferation) comprising orallyadministering a cytidine analog provided herein (e.g., 5-azacytidine)for release substantially in the stomach, wherein the methods avoidadverse effects associated with administering a cytidine deaminaseinhibitor (e.g., THU) by not co-administering the cytidine deaminaseinhibitor with the cytidine analog. In particular embodiments, acytidine deaminase inhibitor (e.g., THU) is co-administered with thecytidine analog in an amount of, e.g., less than about 500 mg/d, lessthan about 200 mg/d, less than about 150 mg/d, less than about 100 mg/d,less than about 50 mg/d, less than about 25 mg/d, less than about 10mg/d, less than about 5 mg/d, less than about 1 mg/d, or less than about0.1 mg/d.

In certain embodiments, methods provided herein comprise treating adisorder provided herein, including a hematologic disorder, byadministering an oral dosage form comprising a cytidine analog to asubject in need thereof. In particular embodiments, oral dosage formsprovided herein comprising 5-azacytidine are used to treat subjectshaving hematologic disorders. Hematologic disorders include, e.g.,abnormal growth of blood cells which can lead to dysplastic changes inblood cells and hematologic malignancies such as various leukemias.Examples of hematologic disorders include, but are not limited to, acutemyeloid leukemia (AML), acute promyelocytic leukemia (APML), acutelymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML),chronic lymphocytic leukemia (CLL), myelodysplastic syndromes (MDS), andsickle cell anemia, among others. Other disorders that can be treatedusing the methods provided herein include, e.g., multiple myeloma (MM)and non-Hodgkin's lymphoma (NHL).

In certain embodiments, methods provided herein comprise treating AML byadministering an oral dosage form comprising a cytidine analog to asubject in need thereof. AML is the most common type of acute leukemiathat occurs in adults. Several inherited genetic disorders andimmunodeficiency states are associated with an increased risk of AML.These include disorders with defects in DNA stability, leading to randomchromosomal breakage, such as Bloom's syndrome, Fanconi's anemia,Li-Fraumeni kindreds, ataxia-telangiectasia, and X-linkedagammaglobulinemia.

In certain embodiments, methods provided herein comprise treating APMLby administering an oral dosage form comprising a cytidine analog to asubject in need thereof. APML represents a distinct subgroup of AML.This subtype is characterized by promyelocytic blasts containing the 15;17 chromosomal translocation. This translocation leads to the generationof the fusion transcript comprised of the retinoic acid receptor and asequence PML.

In certain embodiments, methods provided herein comprise treating ALL byadministering an oral dosage form comprising a cytidine analog to asubject in need thereof. ALL is a heterogenerous disease with distinctclinical features displayed by various subtypes. Reoccurring cytogeneticabnormalities have been demonstrated in ALL. The most common cytogeneticabnormality is the 9; 22 translocation. The resultant Philadelphiachromosome represents poor prognosis of the subject.

In certain embodiments, methods provided herein comprise treating CML byadministering an oral dosage form comprising a cytidine analog to asubject in need thereof. CML is a clonal myeloproliferative disorder ofa pluripotent stem cell. CML is characterized by a specific chromosomalabnormality involving the translocation of chromosomes 9 and 22,creating the Philadelphia chromosome. Ionizing radiation is associatedwith the development of CML.

In certain embodiments, methods provided herein comprise treating MDS byadministering an oral dosage form comprising a cytidine analog to asubject in need thereof. In certain embodiments, MDS includes one ormore of the following myelodysplastic syndrome subtypes: refractoryanemia, refractory anemia with ringed sideroblasts (if accompanied byneutropenia or thrombocytopenia or requiring transfusions), refractoryanemia with excess blasts, refractory anemia with excess blasts intransformation, and chronic myelomonocytic leukemia. In certainembodiments, the MDS is higher-risk MDS. In certain embodiments, themethods provided herein comprise administering an oral dosage formcomprising a cytidine analog to a subject in need thereof to increasethe survival (e.g., prolong the life) of a subject with MDS.

Non-Hodgkin lymphoma (NHL) is any of a large group of cancers oflymphocytes. These types can be divided into aggressive (fast-growing)and indolent (slow-growing) types, and they can be formed from eitherB-cells or T-cells. B-cell NHLs include Burkitt lymphoma, chroniclymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), diffuse largeB-cell lymphoma, follicular lymphoma, immunoblastic large cell lymphoma,precursor B-lymphoblastic lymphoma, and mantle cell lymphoma. T-cellNHLs include mycosis fungoides, anaplastic large cell lymphoma, andprecursor T-lymphoblastic lymphoma.

In certain embodiments, methods provided herein comprise treating NHL byadministering an oral dosage form comprising a cytidine analog to asubject in need thereof. Non-Hodgkin's Lymphomas (NHL) represent aheterogeneous group of malignancies of the lymphoid system. According tothe WHO classification of hematological and lymphoid tumors, thesediseases are classified as B-cell and T-cell neoplasms. B-cell lymphomasaccount for about 90% of all lymphomas, and the two most commonhistological disease entities are follicular lymphoma and diffuse largeB-cell lymphoma. Approximately 55,000 to 60,000 new cases of NHL arediagnosed annually in the U.S. See, e.g., Ansell, S. M., et al., MayoClin. Proc., 2005, 80(8):1087-97.

In one embodiment, methods provided herein comprise treating diffuselarge B-cell lymphoma, which comprises administering to a human havingdiffuse large B-cell lymphoma a therapeutically effective amount of5-azacytidine, or a pharmaceutically acceptable salt, solvate or hydratethereof, and optionally administering therapeutically effective amountsof one or more additional active agents. In one embodiment, the diffuselarge B-cell lymphoma is previously untreated diffuse large B-celllymphoma.

In one embodiment, methods provided herein comprise treating follicularlymphoma which comprises administering to a human having follicularlymphoma a therapeutically effective amount of 5-azacytidine, or apharmaceutically acceptable salt, solvate or hydrate thereof, andoptionally administering therapeutically effective amounts of one ormore additional active agents. In one embodiment, the follicularlymphoma is Grade 3B follicular lymphoma. In one embodiment, thefollicular lymphoma is previously untreated Grade 3B follicularlymphoma.

In one embodiment, methods provided herein comprise treating mantel celllymphoma which comprises administering to a human having mantel celllymphoma a therapeutically effective amount of 5-azacytidine, or apharmaceutically acceptable salt, solvate or hydrate thereof, andoptionally administering therapeutically effective amounts of one ormore additional active agents. In one embodiment, the mantel celllymphoma is previously untreated mantel cell lymphoma.

In one embodiment, one or more additional active agents compriserituximab, cyclophosphamide, doxorubicin, vincristine, prednisone, or acombination thereof. In one embodiment, one or more additional activeagents comprise rituximab, cyclophosphamide, doxorubicin, vincristine,and prednisone.

In one embodiment, methods provided herein comprise treating diffuselarge B-cell lymphoma, which comprises administering to a human havingdiffuse large B-cell lymphoma therapeutically effective amounts of5-azacytidine, rituximab, cyclophosphamide, doxorubicin, vincristine,and prednisone.

In one embodiment, methods provided herein comprise treating follicularlymphoma, which comprises administering to a human having follicularlymphoma therapeutically effective amounts of 5-azacytidine, rituximab,cyclophosphamide, doxorubicin, vincristine, and prednisone.

In one embodiment, methods provided herein comprise treating mantel celllymphoma, which comprises administering to a human having mantel celllymphoma therapeutically effective amounts of 5-azacytidine, rituximab,cyclophosphamide, doxorubicin, vincristine, and prednisone.

In one embodiment, the 5-azacytidine and the one or more additionalactive agents are administered cyclically. In one embodiment, a cyclecomprises 21 days. In one embodiment, the one or more additionaltherapeutic agents are administered on day 1 of a 21 day cycle. In oneembodiment, 5-azacytidine, rituximab, cyclophosphamide, doxorubicin,vincristine, and prednisone are administered cyclically. In oneembodiment, rituximab, cyclophosphamide, doxorubicin and vincristine areadministered on Day 1, and prednisone is administered on Days 1-5.

In one embodiment, rituximab, cyclophosphamide, doxorubicin andvincristine are administered on Day 1, and prednisone is administeredfor five days at the end of the 21 day cycle.

In one embodiment, the 5-azacytidine is administered orally. In oneembodiment, the 5-azacytidine is administered in the form of a capsuleor tablet. In one embodiment, the 5-azacytidine is administered in atablet in an amount from about 40 mg to about 480 mg. In one embodiment,the 5-azacytidine is administered in a tablet in an amount of about 100mg, 150 mg, 200 mg, 300 mg or 400 mg.

In one embodiment, the diffuse large B-cell lymphoma is relapsed,refractory, or relapsed and refractory diffuse large B-cell lymphoma. Inone embodiment, the follicular lymphoma is relapsed, refractory, orrelapsed and refractory follicular lymphoma. In one embodiment, themantel cell lymphoma is relapsed, refractory, or relapsed and refractorymantel cell lymphoma.

In certain embodiments, methods provided herein comprise treating MM byadministering an oral dosage form comprising a cytidine analog to asubject in need thereof. Multiple myeloma is one of the most commonlydiagnosed hematologic malignancies. In 2007, in the U.S. alone, therewere roughly 20,000 new MM cases and 10,000 deaths due to MM. Thedisease is characterized by, inter alia, an accumulation of malignantplasma cells in the bone marrow, which can lead to the overproduction ofan immunoglobulin, e.g., a monoclonal immunoglobulin G or A. Theseimmunoglobulins, also known as paraproteins, can be detected in theurine and blood of patients with MM. Consequences of MM include anemia,the development of destructive bony lesions, and renal insufficiency.See, e.g., Rao, K. V., American Journal of Health-System Pharmacy, 2007,64(17):1799-1807.

In certain embodiments, methods provided herein comprise treating CLL byadministering an oral dosage form comprising a cytidine analog to asubject in need thereof. Chronic lymphocytic lymphoma (CLL) is amalignancy of mature B lymphocytes and is the most prevalent lymphoidmalignancy in the U.S. The WHO classification of B lymphocytic neoplasmsgroups B cell malignancies according to the presumed normal counterpartof the malignant cells. CLL is diagnosed by immunophenotype analysis oflymphocytes from the blood, bone marrow, or lymph nodes. See, e.g.,Zent, C. S., et al., Current Oncology Reports, 2007, 9:345-52.

Certain embodiments herein provide methods for delivering a cytidineanalog to a subject comprising administering to the subject in needthereof an oral formulation comprising a cytidine analog. In particularembodiments, oral formulations comprise (1) a therapeutically effectiveamount of a cytidine analog; and (2) an optional drug releasecontrolling component capable of releasing the cytidine analogsubstantially in the stomach after a subject ingests the oralformulation comprising the cytidine analog. Certain embodiments hereinprovide a method for enhancing the oral bioavailability of a cytidineanalog in a subject. Certain embodiments herein provide a method ofincreasing the oral bioavailability of a cytidine analog comprisingorally administering a pharmaceutical composition provided herein. Incertain methods provided herein, a pharmaceutical composition providedherein is orally administered to a subject, contacts the biologicalfluids of the subject's body, and is absorbed in the uppergastrointestinal tract, such as, for example, substantially in thestomach.

Certain embodiments herein provide a method of achieving a particularexposure value provided herein by administering an oral formulationcomprising a cytidine analog (e.g., 5-azacytidine) provided herein.Certain embodiments herein provide a method of achieving a particularoral bioavailability value provided herein by administering an oralformulation comprising a cytidine analog (e.g., 5-azacytidine) providedherein. Certain embodiments herein provide a method of achieving aparticular AUC value provided herein by administering an oralformulation comprising a cytidine analog (e.g., 5-azacytidine) providedherein. Certain embodiments herein provide a method of achieving aparticular Cmax value provided herein by administering an oralformulation comprising a cytidine analog (e.g., 5-azacytidine) providedherein. Certain embodiments herein provide a method of achieving aparticular Tmax value provided herein by administering an oralformulation comprising a cytidine analog (e.g., 5-azacytidine) providedherein.

Certain embodiments herein provide methods of treating a conditioninvolving undesirable or uncontrolled cell proliferation byadministering an oral formulation comprising a cytidine analog (e.g.,5-azacytidine) as provided herein. Such conditions include, e.g., benigntumors, various types of cancers such as primary tumors and tumormetastasis, hematological disorders (e.g. leukemia, myelodysplasticsyndrome and sickle cell anemia), restenosis (e.g. coronary, carotid,and cerebral lesions), abnormal stimulation of endothelial cells(arteriosclerosis), insults to body tissue due to surgery, abnormalwound healing, abnormal angiogenesis, diseases that produce fibrosis oftissue, repetitive motion disorders, disorders of tissues that are nothighly vascularized, and proliferative responses associated with organtransplants.

In certain embodiments, cells in a benign tumor retain theirdifferentiated features and do not divide in a completely uncontrolledmanner. A benign tumor may be localized and/or nonmetastatic. Specifictypes of benign tumors that can be treated using the methods,compositions, and formulations provided herein include, e.g.,hemangiomas, hepatocellular adenoma, cavernous hemangioma, focal nodularhyperplasia, acoustic neuromas, neurofibroma, bile duct adenoma, bileduct cystanoma, fibroma, lipomas, leiomyomas, mesotheliomas, teratomas,myxomas, nodular regenerative hyperplasia, trachomas and pyogenicgranulomas.

In certain embodiments, cells in a malignant tumor becomeundifferentiated, do not respond to the body's growth control signals,and/or multiply in an uncontrolled manner. The malignant tumor may beinvasive and capable of spreading to distant sites (metastasizing).Malignant tumors may be divided into two categories: primary andsecondary. Primary tumors arise directly from the tissue in which theyare found. A secondary tumor, or metastasis, is a tumor which isoriginated elsewhere in the body but has now spread to a distant organ.The common routes for metastasis are direct growth into adjacentstructures, spread through the vascular or lymphatic systems, andtracking along tissue planes and body spaces (peritoneal fluid,cerebrospinal fluid, etc.).

Methylation can lead to the silencing of genes critical to cellularcontrol (i.e., epigenetic gene silencing), and can be an early event inthe development of malignant tumors including, e.g., colorectal canceror lung cancer. See, e.g., M. V. Brock et al., N. Engl. J Med., 2008,358(11):1118-28; P. M. Das et al., Mol. Cancer, 2006, 5(28); G. Giffordet al., Clin. Cancer Res., 2004, 10:4420-26; J. G. Herman et al., N.Engl. J. Med., 2003, 349:2042-54; A. M. Jubb et al., J. Pathology, 2001,195:111-34. Accordingly, in certain embodiments, methods herein provideusing oral formulations provided herein to prevent or reverse epigeneticgene silencing, e.g., by reversing abnormal DNA methylation. In specificembodiments, oral formulations provided herein are used for earlyintervention to prevent the development of cancer in patients at risk ofdeveloping cancer, e.g., familial polyposis or lung cancer, wherein acause of the cancer is epigenetic gene silencing. In particularembodiments, such early intervention would be impractical by means otherthan oral administration (e.g., IV or SC administration). In specificembodiments, oral formulations provided herein are used for earlyintervention to prevent the recurrence of cancer in patients at risk forearly relapse, e.g., colorectal cancer or non-small-cell lung cancer. Incertain embodiments, the early intervention is achieved via prolongedoral dosing schedules, using formulations and/or methods as describedherein. Certain embodiments provide methods for administering oralformulations provided herein to reverse the effect of gene silencing,e.g., in patients at risk of gene silencing due to epigenetic changes.In particular embodiments, methods provided herein further compriseadministering an HDAC inhibitor compound (e.g., to restore chromatin toa transcriptionally active configuration after reversing abnormal DNAmethylation). In particular embodiments, the HDAC inhibitor compound isentinostat (SNDX-275; formerly MS-275), an oral HDAC inhibitor that actssynergistically with targeted therapies and is selective forcancer-relevant HDAC isoforms 1, 2, and 3. In particular embodiments, asynergistic effect is achieved by co-administering 5-azacytidine and anHDAC inhibitor (e.g., etinostat) for the treatment of solid tumors(e.g., NSCLC) or hematological malignancies (e.g., MDS, CMMoL, or AML).

In certain embodiments, specific types of cancers or malignant tumors,either primary or secondary, that can be treated using the methods,compositions, and formulations provided herein include, e.g., leukemia,breast cancer, skin cancer, bone cancer, prostate cancer, liver cancer,lung cancer (e.g., non-small-cell lung cancer and small-cell lungcancer), brain cancer, cancer of the larynx, gall bladder, pancreas,rectum, parathyroid, thyroid, adrenal, neural tissue, head and neck,colon, stomach, bronchi, kidneys, basal cell carcinoma, squamous cellcarcinoma of both ulcerating and papillary type, metastatic skincarcinoma, osteo sarcoma, Ewing's sarcoma, veticulum cell sarcoma,myeloma, giant cell tumor, gallstones, islet cell tumor, primary braintumor, acute and chronic lymphocytic and granulocytic tumors, hairy-celltumor, adenoma, hyperplasia, medullary carcinoma, pheochromocytoma,mucosal neuronmas, intestinal ganglioneuromas, hyperplastic cornealnerve tumor, marfanoid habitus tumor, Wilm's tumor, seminoma, ovariantumor, leiomyoma tumor, cervical dysplasia and in situ carcinoma,neuroblastoma, retinoblastoma, medulloblastoma, soft tissue sarcoma,malignant carcinoid, topical skin lesion, mycosis fungoides,rhabdomyosarcoma, Kaposi's sarcoma, osteogenic and other sarcoma,malignant hypercalcemia, renal cell tumor, polycythermia vera,adenocarcinoma, glioblastoma multiforma, leukemias, lymphomas, malignantmelanomas, epidermoid carcinomas, and other carcinomas and sarcomas.

Particular embodiments herein provide using the methods, compositions,and formulations provided herein to treat abnormal cell proliferationdue to, e.g., insults to body tissue during surgery for a variety ofsurgical procedures, including, e.g., joint surgery, bowel surgery, andcheloid scarring. Proliferative responses associated with organtransplantation that may be treated using the methods, compositions, andformulations provided herein include those proliferative responsescontributing to potential organ rejections or associated complications.Specifically, these proliferative responses may occur duringtransplantation of the heart, lung (e.g., non-small-cell lung cancer andsmall-cell lung cancer), liver, kidney, and other body organs or organsystems.

In certain embodiments, the amount of the cytidine analog in theformulations provided herein, the methods of administration thereof, orthe methods of treatment as set forth herein, is a specific dosageamount as provided herein. In certain embodiments, oral azacitidinedosages, methods of administration thereof, or methods of treatment ofat least one condition, including but not limited to MDS and AML, mayrange, e.g., between about 50 mg/m²/day and about 2,000 mg/m²/day,between about 100 mg/m²/day and about 1,000 mg/m²/day, between about 100mg/m²/day and about 500 mg/m²/day, or between about 120 mg/m²/day andabout 250 mg/m²/day. In certain embodiments, particular dosages are,e.g., about 120 mg/m²/day, about 140 mg/m²/day, about 150 mg/m²/day,about 180 mg/m²/day, about 200 mg/m²/day, about 220 mg/m²/day, about 240mg/m²/day, about 250 mg/m²/day, about 260 mg/m²/day, about 280mg/m²/day, about 300 mg/m²/day, about 320 mg/m²/day, about 350mg/m²/day, about 380 mg/m²/day, about 400 mg/m²/day, about 450mg/m²/day, or about 500 mg/m²/day.

In certain embodiments, appropriate biomarkers may be used to determineor predict the effect of the pharmaceutical compositions comprisingcytidine analogs on the disease state and to provide guidance to thedosing schedule. For example, particular embodiments herein provide amethod of determining whether a patient diagnosed with MDS has anincreased probability of obtaining a greater benefit from treatment witha pharmaceutical composition comprising a cytidine analog by assessingthe patient's nucleic acid methylation status. In particularembodiments, the cytidine analog is azacitidine. In particularembodiments, the nucleic acid is DNA or RNA. In particular embodiments,the greater benefit is an overall survival benefit. In particularembodiments, the methylation status is examined in one or more genes,e.g., genes associated with MDS or AML. Specific embodiments involvemethods for determining whether baseline DNA methylation levelsinfluence overall survival in patients with MDS (e.g., higher risk MDS)treated with azacitidine. Specific embodiments provide methods fordetermining whether gene promoter methylation levels influence overallsurvival in patients with MDS (e.g., higher risk MDS).

For example, specific embodiments herein provide methods for evaluatingthe influence of gene methylation on prolonged survival in patients withMDS (e.g., higher risk MDS). In particular embodiments, such evaluationis used to predict overall survival in patients with MDS (e.g., higherrisk MDS), e.g., upon treatment with a pharmaceutical compositioncomprising a cytidine analog, as provided herein. In particularembodiments, such evaluation is used for therapeutic decision-making. Inspecific embodiments, such therapeutic decision-making includes planningor adjusting a patient's treatment, e.g., the dosing regimen, amount,and/or duration of administration of the cytidine analogue.

Certain embodiments provide methods of identifying individual patientsdiagnosed with MDS having an increased probability of obtaining anoverall survival benefit from cytidine analog treatment, using analysisof methylation levels, e.g., in particular genes. In specificembodiments, lower levels of nucleic acid methylation are associatedwith an increased probability of obtaining improved overall survivalfollowing azacitidine treatment. In particular embodiments, theincreased probability of obtaining improved overall survival followingtreatment is at least a 5% greater probability, at least a 10% greaterprobability, at least a 20% greater probability, at least a 30% greaterprobability, at least a 40% greater probability, at least a 50% greaterprobability, at least a 60% greater probability, at least a 70% greaterprobability, at least an 80% greater probability, at least a 90% greaterprobability, at least at least a 100% greater probability, at least a125% greater probability, at least a 150% greater probability, at leasta 175% greater probability, at least a 200% greater probability, atleast a 250% greater probability, at least a 300% greater probability,at least a 400% greater probability, or at least a 500% greaterprobability of obtaining improved overall survival following treatment,e.g., using a pharmaceutical composition comprising a cytidine analog asprovided herein. In particular embodiments, the greater probability ofobtaining improved overall survival following treatment is a greaterprobability as compared to the average probability of a particularcomparison population of patients diagnosed with MDS. In specificembodiments, the comparison population is a group of patients classifiedwith a particular myelodysplastic subtype, as described herein. In oneembodiment, the comparison population consists of patients having higherrisk MDS. In particular embodiments, the comparison population consistsof a particular IPSS cytogenetic subgroup.

In particular embodiments, nucleic acid (e.g., DNA or RNA)hypermethylation status may be determined by any method known in theart. In certain embodiments, DNA hypermethylation status may bedetermined using the bone marrow aspirates of patients diagnosed withMDS, e.g., by using quantitative real-time methylation specific PCR(“qMSP”). In certain embodiments, the methylation analysis may involvebisulfite conversion of genomic DNA. For example, in certainembodiments, bisulfite treatment of DNA is used to convertnon-methylated CpG sites to UpG, leaving methylated CpG sites intact.See, e.g., Frommer, M., et al., Proc. Nat'l Acad. Sci. USA 1992,89:1827-31. Commercially available kits may be used for such bisulfitetreatment. In certain embodiments, to facilitate methylation PCR,primers are designed as known in the art, e.g., outer primers whichamplify DNA regardless of methylation status, and nested primers whichbind to methylated or non-methylated sequences within the regionamplified by the first PCR. See, e.g., Li et al., Bioinformatics 2002,18:1427-31. In certain embodiments, probes are designed, e.g., probeswhich bind to the bisulfite-treated DNA regardless of methylationstatus. In certain embodiments, CpG methylation is detected, e.g.,following PCR amplification of bisulfite-treated DNA using outerprimers. In certain embodiments, amplified product from the initial PCRreaction serves as a template for the nested PCR reaction usingmethylation-specific primers or non-methylation-specific primers. Incertain embodiments, a standard curve is established to determine thepercentage of methylated molecules in a particular sample. Methods fordetecting nucleic acid methylation (e.g., RNA or DNA methylation) areknown in art. See, e.g., Laird, P. W., Nature Rev. Cancer 2003,3:253-66; Belinsky, S. A., Nature Rev. Cancer 2004, 4:1-11.

In certain embodiments, statistical analyses are performed to assess theinfluence of particular methylation levels with the potential benefit oftreatment with a particular pharmaceutical composition comprising acytidine analog. In certain embodiments, the influence of methylation onoverall survival is assessed, e.g., using Cox proportional hazardsmodels and Kaplan-Meier (KM) methodology.

In certain embodiments, any gene associated with MDS and/or AML may beexamined for its methylation status in a patient. Particular genesinclude, but are not limited to, CKDN2B (p15), SOCS1, CDH1 (E-cadherin),TP73, and CTNNA1 (alpha-catenin). Particular genes associated with MDSand/or AML, which would be suitable for use in the methods disclosedhere, are known in the art.

1. Methods Comprising Co-Administering One or More AdditionalTherapeutic Agents with the Oral Formulations Disclosed Herein

Certain embodiments herein provide methods of treating diseases ordisorders disclosed herein (e.g., diseases or disorders involvingabnormal cell proliferation), wherein the methods compriseco-administering an oral formulation disclosed herein (such as, forexample, an oral formulation comprising 5-azacytidine) with one or moreadditional therapeutic agents (such as, for example, a cancertherapeutic agent) to yield a synergistic therapeutic effect. Particularco-administered therapeutic agents useful in the methods disclosedherein are disclosed throughout the specification. In particularembodiments, the additional therapeutic agent is co-administered in anamount that is a therapeutically effective amount. In particularembodiments, the additional therapeutic agent is co-administered in aseparate dosage form from the cytidine analog dosage form with which itis co-administered. In particular embodiments, the additionaltherapeutic agent is co-administered in a dosage form (e.g., a singleunit dosage form) together with the cytidine analog with which it isco-administered. In such cases, the cytidine analog (e.g., azacitidine)and the additional therapeutic agent may be co-formulated together inthe same dosage form using methods of co-formulating activepharmaceutical ingredients, including methods disclosed herein andmethods known in the art.

In one embodiment, provided herein is a method of treating diffuse largeB-cell lymphoma or follicular lymphoma, which comprises administering toa human having diffuse large B-cell lymphoma or follicular lymphoma atherapeutically effective amount of 5-azacytidine, or a pharmaceuticallyacceptable salt, solvate or hydrate thereof, and administeringtherapeutically effective amounts of one or more additional activeagents.

In one embodiment, provided herein is a method of treating mantel celllymphoma, which comprises administering to a human having mantel celllymphoma a therapeutically effective amount of 5-azacytidine, or apharmaceutically acceptable salt, solvate or hydrate thereof, andadministering therapeutically effective amounts of one or moreadditional active agents.

In one embodiment, one or more additional active agents compriserituximab, cyclophosphamide, doxorubicin, vincristine, prednisone, or acombination thereof. In one embodiment, additional active agentscomprise rituximab, cyclophosphamide, doxorubicin, vincristine, andprednisone.

In one embodiment, the diffuse large B-cell lymphoma is previouslyuntreated diffuse large B-cell lymphoma.

In one embodiment, the follicular lymphoma is Grade 3B follicularlymphoma. In one embodiment, the follicular lymphoma is previouslyuntreated Grade 3B follicular lymphoma.

In one embodiment, the mantel cell lymphoma is previously untreatedmantel cell lymphoma.

In one embodiment, the 5-azacytidine and the one or more additionalactive agents are administered cyclically. In one embodiment, a cyclecomprises 21 days. In one embodiment, the one or more additionaltherapeutic agents are administered on day 1 of a 21 day cycle.

In one embodiment, the 5-azacytidine is administered until diseaseprogression or unacceptable toxicity.

In one embodiment, the 5-azacytidine is administered orally. In oneembodiment, the 5-azacytidine is administered in the form of a capsuleor tablet. In one embodiment, the 5-azacytidine is administered in atablet in an amount from about 40 mg to about 480 mg. In one embodiment,the 5-azacytidine is administered in a tablet in an amount of about 100mg, 150 mg, 200 mg, 300 mg or 400 mg.

In one embodiment, provided herein is a method of treating diffuse largeB-cell lymphoma, which comprises administering to a human having diffuselarge B-cell lymphoma therapeutically effective amounts of5-azacytidine, rituximab, cyclophosphamide, doxorubicin, vincristine,and prednisone.

In one embodiment, provided herein is a method of treating follicularlymphoma, which comprises administering to a human having follicularlymphoma therapeutically effective amounts of 5-azacytidine, rituximab,cyclophosphamide, doxorubicin, vincristine, and prednisone.

In one embodiment, provided herein is a method of treating mantel celllymphoma, which comprises administering to a human having mantel celllymphoma therapeutically effective amounts of 5-azacytidine, rituximab,cyclophosphamide, doxorubicin, vincristine, and prednisone.

In one embodiment, 5-azacytidine, rituximab, cyclophosphamide,doxorubicin, vincristine, and prednisone are administered cyclically. Inone embodiment, rituximab, cyclophosphamide, doxorubicin and vincristineare administered on Day 1, and prednisone is administered on Days 1-5.In one embodiment, rituximab, cyclophosphamide, doxorubicin andvincristine are administered on Day 1 of a 21 day cycle, and prednisoneis administered on Days 1-5 of a 21 day cycle.

In one embodiment, rituximab, cyclophosphamide, doxorubicin andvincristine are administered on Day 1, and prednisone is administeredeach day for five days at the end of the 21 day cycle. In oneembodiment, rituximab, cyclophosphamide, doxorubicin and vincristine areadministered on Day 1 of a 21 day cycle, and prednisone is administeredon days 1-5 following the 21 day cycle.

Incorporation By Reference

All disclosures (e.g., patents, publications, and web pages) referencedthroughout this specification are incorporated by reference in theirentireties. In addition, the following disclosures are also incorporatedby reference herein in their entireties: (1) 2008 ASCO poster abstractby B. S. Skikne, M. R. Ward, A. Nasser, L. Aukerman, G. Garcia-Manero;and (2) G. Garcia-Manero, M. L. Stoltz, M. R. Ward, H. Kantarjian, andS. Sharma, Leukemia, 2008, 22, 1680-84.

VII. EXAMPLES A. Example 1

5-Azacytidine tablets were manufactured using direct tablet compressionfollowed by optional seal film-coating and/or enteric film-coating, asdescribed below. Table 3 lists the excipients used in each of the tabletformulations. Table 4 describes the formula composition of the tabletsusing weights. Table 5 describes the formula composition of the tabletsusing percentages.

Formulation 1 was manufactured without the seal-coating step, which mayhave resulted in an enteric coat that contained a “leaky” entericcoating. Talc was only used in the enteric coating suspension forFormulation 1.

Except for Formulation 1, a common blend with 20% drug load of5-azacytidine was used to manufacture all tablets. Vitamin E TPGS(d-alpha-tocopheryl polyethylene glycol 1000 succinate) was added tocertain of the formulations to enhance absorption of 5-azacytidine.Vitamin E TPGS was not used in Formulation 6.

Tablets were manufactured using the process described FIG. 1, except forFormulation 1 (which did not undergo the seal-coating step).Formulations 3 and 6 did not undergo the enteric film-coating step, andFormulation 6 did not contain Vitamin E TPGS. The process is generallydescribed as follows:

Mannitol, silicified microcrystalline cellulose, crospovidone, magnesiumstearate and azacitidine were individually screened to ensurede-aggregation of any agglomerates. Vitamin E TPGS was melted in astainless steel vessel to which was then added a portion of thesilicified microcrystalline cellulose (not done in Formulation 6). TheVitamin E TPGS-silicified microcrystalline cellulose mixture was allowedto cool and then screened. Azacitidine, Vitamin E TPGS—silicifiedmicrocrystalline cellulose mix, remaining silicified microcrystallinecellulose, mannitol and crospovidone were mixed in a V-blender.Magnesium stearate was added to the V-blender followed by additionalmixing. The resulting blend was compressed into tablets using standardconcave tooling.

Hydroxypropyl cellulose was dispersed into ethanol. The hydroxypropylcellulose preparation was used to spray coat the tablet cores to prepareseal coated tablets.

EUDRAGIT and triethyl citrate were dispersed into an isopropanol—acetonemixed solvent system. EUDRAGIT—triethyl citrate preparation was used tospray coat the seal coated tablet.

TABLE 3 Components of Azacitidine Tablets Quality Component FunctionStandard Azacitidine API In-House Mannitol Bulking Agent USP SilicifiedMicrocrystalline Cellulose Binding Agent NF d-alpha-tocopherylpolyethylene glycol Permeation NF 1000 succinate (Vitamin E TPGS)Enhancer Polyvinyl Polypyrrolidone (Crospovidone) Disintegrant NFMagnesium Stearate Lubricant NF Hydroxypropyl Cellulose Seal Film CoatNF Ethanol ^(a) Coating Solvent USP Methacrylic Acid Copolymer (EudragitEnteric NF S100, Eudragit LIDO-55 or Eudragit Film Coat L100) TriethylCitrate Plasticizer NF Talc Anti-Caking USP Isopropanol ^(a) CoatingSolvent USP Acetone Coating Solvent NF ^(a) Removed during processing(used as solvent for film-coating polymers).

TABLE 4 Formula Composition of Azacitidine Tablets (Weight) Quantity perUnit Tablet (mg) Formu- Formu- Formu- Formu- Formu- Formu- lation #1lation #2 lation #3 lation #4 lation #5 lation #6 Leaky Enteric-Immediate Enteric- Enteric- Immediate coating coated Release coatedcoated Release Component (pH > 7.0) (pH > 7.0) w/vitamin E (pH > 5.0)(pH > 5.5) w/o vitamin E Azacitidine^(a) 20.0 20.0 60.0 60.0 60.0 60.0Mannitol, USP 59.7 43.2 129.6 129.6 129.6 135.6 Silicified 13.9 30.090.0 90.0 90.0 90.0 Microcrystalline Cellulose, NF Crospovidone, NF 2.83.0 9.0 9.0 9.0 9.0 Magnesium Stearate, 1.6 1.8 5.4 5.4 5.4 5.4 NFVitamin E TPGS, 2.0 2.0 6.0 6.0 6.0 0 NF Core Tablet Total 100.0 100.0300.0 300.0 300.0 300.0 Hydroxypropyl N/A 4.0 12.0 12.0 12.0 12.0Cellulose, NF Ethanol ^(b) N/A — — — — — Seal-Coated Tablet N/A 104.0312.0 312.0 312.0 312.0 Total Eudragit S-100 3.7-5.9 7.0-8.0 N/A N/A N/AN/A Eudragit L 100-55 N/A N/A N/A 21.8-25.0 N/A N/A Eudragit L 100 N/AN/A N/A N/A 28.1-31.2 N/A Triethyl Citrate 0.3-0.5 1.0-2.0 N/A 3.0-6.03.0-6.0 N/A Talc 1.0-1.6 N/A N/A N/A N/A N/A Isopropanol ^(b) — — N/A —— N/A Acetone ^(b) — — N/A — — N/A Total Theoretical 106.5 113.0 312.0335.4 341.64 312.0 Weight ^(a)Assuming 100% purity. ^(b) Removed duringprocessing.

TABLE 5 Formula Composition of Azacitidine Tablets (Percent) Quantityper Unit Tablet (mg) Formu- Formu- Formu- Formu- Formu- Formu- lation #1lation #2 lation #3 lation #4 lation #5 lation #6 Leaky Enteric-Immediate Enteric Enteric Immediate coating coated Release coated coatedRelease Component (pH > 7.0) (pH > 7.0) w/vitamin E (pH > 5.0) (pH >5.5) w/o vitamin E Azacitidine ^(a) 20.0 20.0 20.0 20.0 20.0 20.0Mannitol, USP 59.7 43.2 43.2 43.2 43.2 45.2 Silicified 13.9 30.0 30.030.0 30.0 30.0 Microcrystalline Cellulose, NF Crospovidone, NF 2.8 3.03.0 3.0 3.0 3.0 Magnesium Stearate, 1.6 1.8 1.8 1.8 1.8 1.8 NF Vitamin ETPGS, 2.0 2.0 2.0 2.0 2.0 0.0 NF Core Tablet Total 100.0 100.0 100.0100.0 100.0 100.0 Hydroxypropyl N/A 4.0 4.0 4.0 4.0 4.0 Cellulose, NFEthanol ^(b) N/A — — — — — Seal-Coated Tablet 104.0 104.0 104.0 104.0104.0 Total Eudragit S-100 3.7-5.9 7.0-8.0 N/A N/A N/A N/A Eudragit L100-55 N/A N/A N/A 7.0-8.0 N/A N/A Eudragit L 100 N/A N/A N/A N/A 9.0-10.0 N/A Triethyl Citrate 0.3-0.5 1.0-2.0 N/A 1.0-2.0 1.0-2.0 N/ATalc 1.0-1.6 N/A N/A N/A N/A N/A Isopropanol ^(b) — — N/A — — — Acetone^(b) — — N/A — — ^(a) Assuming 100% purity. ^(b) Removed duringprocessing

B. Example 2

Studies were performed to evaluate the effect of aqueous film coating onhydrolytic degradation of azacitidine. Azacitidine tablets werefilm-coated using aqueous-based solvents without affecting levels ofdegradation. As demonstrated in Table 6, significant levels ofazacitidine degradation products were not observed after aqueous filmcoating.

TABLE 6 Effect of Aqueous Film Coating on Azacitidine Uncoated Test CoreTablet Coated Tablet Assay (% Label Claim) Ave = 103.1 Ave = 99.6Related Substances (% Area) N-Formylguanylribosylurea 0.2 0.1Guanylribosylurea 0.7 0.7 Unspecified ND ND Total 0.9 0.8 MoistureContent (% w/w) NMT 2.5 2.2 ND = Not detected; NMT = No more than

C. Example 3

As described in Example 1, the following six formulations, described inTable 7 and elsewhere in the present specification, were prepared andused in clinical studies as described in the Examples below:

TABLE 7 Formulations of Azacitidine used in clinical studies FormulationAzacitidine in Number Formulation Description #1 20 mg “Leaky”enteric-coated tablet #2 20 mg Enteric-coated tablet, core sealed #3 60mg Seal-coated, immediate release tablet with vitamin E #4 60 mg Entericfilm-coated tablet, target dissolution at pH > 5.5 #5 60 mg Entericfilm-coated tablet, target dissolution at pH > 6.0 #6 60 mg Seal-coated,immediate release tablet without vitamin E

D. Example 4

In a multiple dose escalation study (MTD study; CL005), patients withMDS or AML were selected (Selection criteria: ECOG PS 0-2, adequateorgan function, age >18 years). The patients were dosed with multiple28-day cycles of azacitidine. The study had a 3+3 design. During Cycle1, all patients were dosed subcutaneously with azacitidine at 75 mg/m²×7days. During subsequent cycles (dosing on Day 1-7 for each cycle), thepatients were dosed orally with azacitidine at doses listed in Table 8.PK data were collected during Cycles 1 and 2 on Day 1 and 7, and duringCycles 4, 5, and 7, on Day 7. PD data were collected during each cycle,and hematological responses and/or improvement rates were assessed foreach treatment cycle to determine biologically active dose (BAD). Todate, seven cohorts of patients (3 subjects/cohort) have been studiedand none of the patients have shown dose limited toxicity (DLT). Theoral dose and formulation used for each cohort are listed in Table 8.

TABLE 8 Oral Azacitidine Doses and Formulations Subject Demographics #Subjects Cohort # Oral (Patient No. - Treated/Evaluable # SubjectsDosage Formulation gender, age, dx) for DLT with DLT Cohort 1Formulation 02001 - M, 78, MDS 3/3 0 120 mg #2 (20 mg 02002 - M, 66, MDSRAEB-2 tablets) 04001 - M, 56, MDS RAEB-1 Cohort 2 Formulation 02003 -M, 73, AML 4/3 0 120 mg #1 (20 mg 02004 - M, 61, MDS tablets) 04002 - M,73, MDS RAEB-1 02005 - M, 66, MDS RAEB-1 Cohort 3 Formulation 04004 - F,70, AML 3/3 0 180 mg #1 (20 mg 02006 - M, 61, AML tablets) 03001 - F,70, MDS RAEB-2 Cohort 4 Formulation 02007 - M, 76, CMML 3/3 0 240 mg #3(60 mg 02008 - M, 80, MDS RAEB-1 tablets) 02009 - M, 83, MDS RAEB-2Cohort 5 Formulation 04005 - M, 68, MDS RCMD 3/3 0 300 mg #3 (60 mg02011 - M, 92, MDS RAEB-1 tablets) 02012 - M, 62, MDS RCMD Cohort 6Formulation 02013 - F, 66, MDS RAEB-1 3/3 0 360 mg #3 (60 mg 03002 - M,65, MDS RAEB-1 tablets) 01001 - F, 63, MDS RCMD Cohort 5 Formulation01002 - M, 70, MDS RARS  2/0* 0 480 mg #3 (60 mg 01003 - F, 75, MDS RCMDtablets) *Cycle 2 ongoing

PK profiles for Cycle 1, following 75 mg/m² SC dose of azacitidine, arepresented in FIG. 2. Pharmacokinetic parameters calculated fromazacitidine plasma concentrations following SC doses at 75 mg/m² arepresented in Table 9.

TABLE 9 PK parameters from Cycle 1, following SC doses at 75 mg/m²AUC(0-t) AUC(0-inf) Cmax Tmax Lambda_z t½ Cloral Vdoral (ng*hr/mL)(ng*hr/mL) (ng/mL) (hr) (1/hr) (hr) (L/hr) (L) Day 1 Mean (n = 18) 11351170 741 0.49 0.58 1.53 143 318 SD 514 533 293 0.27 0.29 0.80 53 223Minimum 505 538 224 0.23 0.22 0.61 45 90 Median 991 1030 674 0.50 0.561.24 156 265 Maximum 2821 2950 1310 1.08 1.14 3.15 253 788 CV % 45 46 3954 49 52 37 70 Day 7 Mean (n = 18) 1135 1210 697 0.51 0.62 1.73 133 368SD 477 463 252 0.17 0.39 1.28 43 376 Minimum 510 686 254 0.25 0.16 0.4748 98 Median 1020 1116 716 0.50 0.55 1.26 148 162 Maximum 2718 2783 10501.00 1.49 4.30 223 1383 CV % 42 38 36 34 62 74 33 102

Plasma PK profiles following SC (75 mg/m²) and various PO doses arecompared and presented in FIG. 3. An increase in oral dose did notresult in dose-proportional increase in exposure of azacitidine.

Methylation PD data in cycles 1 and 2, from blood (PBL) and bone marrow(BM) samples, were obtained. The PD data collected from individualpatients from Cohort 4 (Formulation #3, oral dose 240 mg) are presentedin FIGS. 4A-4D and FIGS. 5A-5D.

Subject number 02004 of cohort 2 (61-year-old male with MDS, MDACC) wastreated with a SC cycle of azacitidine, followed by initial oral dosesof 120 mg azacitidine (Formulation #1). The patient received oral dosesof 120 mg×7d azacitidine as in Formulation #1 during Cycles 2-6,followed by oral doses of 180 mg×7d azacitidine during Cycles 7-12. Inthis patient, following a 75 mg/m² SC dose of azacitidine, the AUC valuewas 1000 ng*hr/mL. Following a 180 mg oral dose of azacitidine, the AUCvalue was 330 ng*hr/mL, approximately 33% of the exposure observed forthe SC dose (oral bioavailability=30%).

The PD response data from patient 02004 is presented in FIGS. 6A-6D.Platelets (K/uL), Hgb (g/dL), ANC (K/uL), and Relative BM Blast (%) areplotted vs. sampling dates over the course of the study. The patientdemonstrated a morphologic complete response (CR).

For patient 02004, Hgb (10.8 g/dL at ascreening, 11.1 g/dL at Day 1),Platelets (140 K/uL at both screening and Day 1), ANC (1.46 K/uL atscreening and 1.12 K/uL at Day 1), and BM Blast (2%) values at baselineand Day 1 were above normal or close to normal. This patient had notransfusion (RBC or PLT) prior to enrollment into the study and to dayrequired no transfusions (RBC or PLC) during the study. Per IWG 2006criteria, the patient achieved complete response (CR) (from Days 45-74satisfying all CR criteria for 28 consecutive days). The patientachieved morphologic complete response per the IWG AML criteria.However, with regard to the ANC condition for the IWG 2000 CR criteria,the patient did not meet the criteria for a complete response (3 daysshort of the duration requirement of 56 consecutive days).

For patient 02007, as shown in FIGS. 5A and 5C, Grade 4 thrombocytopeniaand neutropenia developed during the first cycle of treatment withsubcutaneous azacitidine when given at 75 mg/m² for 7 days. The onset ofthe cytopenias occurred between days 14 and 21 at time points consistentwith the existing safety profile of azacitidine when administered 75mg/m² for 7 days as a SC injection. In contrast, the administration oforal azacitidine starting with cycle 2 did not result in grade 3 or 4cytopenias yet still produced an increase in platelets above thebaseline levels. This data supports, e.g., the conclusion that certainoral dosage forms provided herein permit the delivery of azacitidine atlower doses over a more prolonged period of time, and that certain oraldosage forms provided herein alter the safety profile of the cytidineanalog.

Assessment of IWG criteria for certain patients in the MTD study ispresented below in Table 10. The data demonstrate, inter alia, patientimprovement following administration of azacitidine formulated forrelease substantially in the stomach.

TABLE 10 MTD Study; Assessment of IWG Criteria Patient No. IWGAssessment 02004 Fairly healthy at baseline: hgb (11.1 g/dL Cycle 1, Day1); PLT (140 K/μL Cycle 1, Day 1); ANC (1.12 K/μL at Cycle 1, Day 1); BMblasts (2%) values at baseline above normal or close to normal CR perIWG 2006 (Days 45-98) Morphologic CR per the IWG AML criteria (Diagnosisis MDS) 02007 HI-P major improvement per IWG 2000 (Days 35-202)Morphologic CR per the revised IWG AML criteria on Days 43-188 and onsome other days (ANC = 1.89 K/μL, but normal at BL = 2.99 and 1.68; PLT= 314 K/μL; BM = 2, but normal at BL = 3) (Diagnosis is CMML) 02008 HI-Pmajor improvement per IWG 2000 (Days 34-110) 02009 Marrow CR (Days7-111+) per IWG 2006 02011 Marrow CR (Days 7-177+) per IWG 2006Morphologic CR per the revised IWG AML criteria on Day 21 (ANC = 1.18K/μL; PLT = 119 K/μL, but normal at BL = 162 & 194; BM = 3) (Diagnosisis MDS)

Immediate release oral formulations comprising azacitidine demonstratedbioavailability in patients. Observations thus far suggest positiveclinical activity in patients treated with oral azacitidineformulations. No safety issues have thus far been observed with thedoses and schedules described above.

E. Example 5

An oral azacitidine clinical study, referred to as the Rapid AzaClinical Evaluation (RACE) study (CL008), was performed; a summary ofthe study design is depicted in FIG. 7. Several oral formulations wereevaluated in this study. A “3+7” cohort of patients was enrolled in thestudy, i.e., three patients were initially tested per formulation, andthe cohorts could increase in size up to ten patients. Cohorts wereenrolled in parallel. PK data was collected periodically, as indicatedin Table 11.

TABLE 11 RACE Study - PK Study Design; PK Cycle 1, Days 1, 3, 5, 15, 17& 19, and Cycle 2, Day 7 Treatment Day Dose PK Phase (Cycle 1) Day 1 75mg/m² SC Day 3 ± 1* 180 mg Oral Day 5 ± 1* 360 mg Oral Day 15 ± 1* 75mg/m² SC Day 17 ± 1* Oral dose calculated to achieve approximately 80%exposure relative to the 75 mg/m² SC dose up to a maximum dose of 1,200mg. Day 19 ± 1* Oral dose calculated to achieve approximately 120%exposure relative to the 75 mg/m² SC dose up to a maximum dose of 1,200mg. Treatment Phase (Cycles 2-7) Days 1-7 Oral dose calculated toachieve approximately 100% exposure relative to the 75 mg/m² SC dose upto a maximum dose of 1,200 mg. *Dose administered ±1 day, as long atleast 48 hours between doses

Results for Formulation #4:

The plasma PK profile for one subject who received Formulation 4 (i.e.,enteric film-coated tablets for release in upper gastrointestinalregion) is depicted in FIG. 8. Values for AUC (0-t) (ng*hr/mL) were asfollows: SC administration of 75 mg/m² (124 mg)=2390 (day 1) and 2440(day 15); Oral administration of 180 mg=234; Oral administration of 360mg=197; and Oral administration of 1200 mg=66.5 (day 17) and 297 (day19). Tmax for oral administration was reached between 2.5 hr and 3.0 hr.A linear increase of exposure (AUC0-inf) was not observed following 180,360, and 1200 mg oral doses. Relative oral bioavailability rangedbetween 0.8 to 6.7%.

Results for Formulation #6:

The plasma PK profile for one subject who received Formulation 6 (i.e.,seal-coated immediate release tablets without vitamin E) is depicted inFIG. 9. Values for AUC(0-∞) (ng*hr/mL) were as follows: SCadministration of 75 mg/m² (120 mg)=1720 (day 1) and 1640 (day 15); Oraladministration of 180 mg=231; Oral administration of 360 mg=280; andOral administration of 1200 mg=543 (day 17) and 467 (day 19). Tmax fororal administration was reached between 0.5 hr and 1.0 hr. A linearincrease of exposure (AUC 0-∞) was observed following 180, 360, and 1200mg oral doses, although the increase was not proportional with dose.Following the 1200 mg oral doses, AUC was approximately 30% of AUCfollowing SC dosing (i.e., about 500 and about 1,700, respectively).

Data from this study indicated that azacitidine was absorbed followingoral administration of immediate release formulations comprisingazacitidine. As compared to SC administration of azacitidine, theimmediate release azacitidine formulations provided a superior percentexposure (e.g., about 30%) than enteric-coated azacitidine formulations.Data supports single or multiple daily dosing of oral azacitidine.

F. Example 6

Based on data from clinical studies involving different azacitidineformulations and dosage amounts, plots were prepared comparing differentformulations with respect to, e.g., their resulting PK profiles, AUCvalues, Cmax values, relative oral bioavailability values, and exposurevalues. Oral formulations involved in the comparisons includeFormulation #3 (“F3”); Formulation #4 (“F4”); and Formulation #6 (“F6”);these oral formulations are described elsewhere herein (e.g., inExamples 1 and 3).

Comparisons of Formulation Nos. 3, 4, and 6

FIG. 10 compares PK profiles (using a linear scale) followingadministration of azacitidine via SC (75 mg/m²; n=18) and oraladministration. For Formulation #3, a total of 360 mg azacitidine wasorally administered (n=6); for Formulation #4, a total of 360 mgazacitidine was orally administered (n=3); for Formulation #6, a totalof 360 mg azacitidine was orally administered (n=5). The plotillustrates immediate release characteristics of Formulations #3 and #6,as compared to Formulation #4, which was enteric coated. FIG. 11provides the same data, plotted on a semi-log scale.

Patients were dosed with azacitidine SC (75 mg/m²) and orally withFormulations #3, #4, or #6 (“F3”; “F4”; and “F6”; described elsewhereherein) with a total of 180 mg, 240 mg, 300 mg, 360 mg, 540 mg, 600 mg,720 mg, 900 mg, 1080 mg, or 1200 mg azacitidine administered perpatient. Results showed that azacitidine is absorbed following oraladministration. As described below, particular values were measured andcompared, including AUC values, Cmax values, relative oralbioavailability values, and exposure values (oral) compared to SC.

FIG. 12 displays AUC values (ng*hr/mL; mean±SD) following azacitidineadministration. FIG. 13 displays Cmax values (ng/mL; mean±SD) followingazacitidine administration. For Formulation #4 (enteric coated), overthe dose range of 180 mg to 1200 mg, an increase in dose did nottranslate into an increase in exposure, and absorption was poor. ForFormulation #3 (immediate release tablets with vitamin E), over the doserange of 180 mg to 1200 mg, an increase in dose translated into anincrease in exposure. For Formulation #6 (immediate release tabletswithout vitamin E), over the dose range of 180 mg to 1200 mg, anincrease in dose translated into an increase in exposure. Tmax forimmediate-release Formulations #3 and #6 were similar: For Formulation#3, median Tmax was 1.1 hr (range 0.5, 2.5 hr); For Formulation #6,median Tmax was 1.0 hr (range 0.5, 3.0 hr).

FIG. 14 displays relative oral bioavailability (%; mean±SD) followingoral dosing with Formulations #3, #4, and #6, at various azacitidinedosage levels. At dosage levels less than or equal to 360 mgazacitidine, Formulation #4 (enteric coated) had a mean relative oralbioavailability of less than 4%. At dosage levels less than or equal to360 mg azacitidine, Formulation #3 (immediate release with vitamin E)had a mean relative oral bioavailability ranging from 11% to 21%. Atdosage levels less than or equal to 360 mg azacitidine, Formulation #6(immediate release without vitamin E) had a mean relative oralbioavailability ranging from 11% to 14%.

FIG. 15 displays exposure (% as compared to SC; mean±SD) following oraldosing with Formulations #3, #4, and #6, at various azacitidine dosagelevels. At dosage levels less than or equal to 360 mg azacitidine,Formulation #4 (enteric coated) had a mean exposure of less than 8%. Atdosage levels less than or equal to 360 mg azacitidine, Formulation #3(immediate release with vitamin E) had a mean exposure ranging from 18%to 37%. At dosage levels less than or equal to 360 mg azacitidine,Formulation #6 (immediate release without vitamin E) had a mean exposureranging from 20% to 31%. As compared to enteric-coated Formulation #4,the immediate-release Formulations #3 and #6 provided superior exposurecompared to SC (about 30% at total dosage amount of 360 mg).

Comparisons of Formulation Nos. 3 and 6

FIG. 16 displays a linear scale profiles of azacitidine plasmaconcentration (ng/ml) versus time (hr) for Formulation #3 and #6 at adosage level of 180 mg (n=6). FIG. 17 displays linear scale profiles ofazacitidine plasma concentration (ng/ml) versus time (hr) forFormulation #3 and #6 at a dosage level of 360 mg (n=6).

FIG. 18 displays a plot of individual (“ind”) and mean azacitidineAUC(0-inf) (ng*hr/ml) versus azacitidine dose (mg) for Formulation #3and #6, with linear regression analysis. Linear regression equations forF3 and F6 are also indicated on the plot. Using those equations, for aselected dose, the expected AUC(0-inf) (ng*hr/ml) were calculated.Calculated values are provided in Table 12.

TABLE 12 Expected AUC(0-inf) Calculated for Formulation #3 and #6Azacitidine AUC(0-inf) (ng*hr/ml) Dose (mg) Formulation #6 Formulation#3 240 263 338 360 296 363 480 328 388 600 361 413 720 393 438 1200 523538 F6 linear regression equation: y = 0.2706 x + 198.19 F3 linearregression equation: y = 0.2079 x + 288.07

FIG. 19 displays a comparison of azacitidine percent relative oralbioavailability (mean±SD) versus azacitidine dose (mg), following dosingwith Formulation #3 or #6, for azacitidine oral dosage amounts including180 mg, 240 mg, 300 mg, 360 mg, 480 mg, 600 mg, 720 mg, 900 mg, 1020 mg,1080 mg, 1140 mg, and 1200 mg. At doses greater than or equal to 1020mg, the mean relative oral bioavailability for Formulation #6 rangedfrom 9% to 14%, and the mean relative oral bioavailability forFormulation #3 ranged from 10% to 21%.

FIG. 20 displays a comparison of azacitidine percent oral exposure ascompared to SC azacitidine dosing (mean±SD) versus azacitidine dose(mg), following oral administration of Formation #3 or #6. Azacitidineoral dosage amounts included 180 mg, 240 mg, 300 mg, 360 mg, 480 mg, 600mg, 720 mg, 900 mg, 1020 mg, 1080 mg, 1140 mg, and 1200 mg. At doseswith n>1, the mean exposures of Formulation #6 and #3, as compared toSC, were similar.

G. Example 7

DNA methylation was employed as a biomarker to monitor responses inpatients treated with azacitidine in the clinical studies describedherein. Analysis was performed with an Infinium Assay (commerciallyavailable from Illumina, Inc., San Diego, Calif.). The Infinium Assaycombined with BeadChips allows large-scale interrogation of variationsin the human genome. For example, the Infinium HumanMethylation27BeadChip enables interrogation of 27,578 CpG loci, covering over 14,000genes. The DNA Methylation Assay protocol included the following steps:(1) bisulfite conversion; (2) DNA amplification; (3) DNA fragmentation;(4) DNA precipitation; (5) DNA hybridization to BeadChip; (6) extensionand staining on BeadChip; and (7) imaging of BeadChip.

The assay for methylation was used to detect methylation status atindividual CpG loci by typing bisulfite-converted DNA. Methylationprotected C from conversion, whereas unmethylated C was converted to T.A pair of bead-bound probes was used to detect the presence of T or C byhybridization followed by single-base extension with a labelednucleotide. Up to twelve samples were profiled in parallel. Blood andbone marrow samples were collected and DNA methylation was analyzed inparallel.

H. Example 8

A study is performed to examine whether baseline DNA and/or RNAmethylation levels influence overall survival (OS) as well as theinteraction between gene promotor methylation levels and treatment(e.g., azacitidine or conventional care regimens (“CCR”)). Methylationis determined for 5 genes previously evaluated in MDS or AML: CDKN2B(p15), SOCS1, CDH1 (E-cadherin), TP73, and CTNNA1 (alpha-catenin), inpre-treatment bone marrow aspirates of patients enrolled in a clinicalstudy using quantitative real-time methylation specific PCR (qMSP). Theinfluence of methylation on OS is assessed using Cox proportionalhazards models and Kaplan-Meier (KM) methodology.

The number of patients (e.g., for azacitidine and CCR) having nucleicacid sufficient for analysis of these 5 genes is determined. Methylationis detected in a specific percentage of patients for CDKN2B, SOCS1,CDH1, TP73, and CTNNA1. Differences in methylation levels between thetreatment arms are determined. The OS benefit for cytidine analog (e.g.,azacitidine) treatment is determined for patients who are positive andnegative for methylation at these 5 genes. It is determined whether thepresence of methylation is associated with improvement in OS in the CCRgroup (prognostic indicator of good outcome). The existence andmagnitude of any effect is compared to the cytidine analog group, whichmay suggest an interaction between DNA and/or RNA methylation andtreatment.

OS improvement is assessed with cytidine analog (e.g., azacitidine)treatment in patients with methylation at any of these 5 genes, and HRof death for methylation is determined. The frequency of methylation ofparticular genes allows for examination of the influence of methylationlevel on OS and treatment effect. For example, for particular genes,lower levels of methylation may be associated with the longest OS andthe greatest OS benefit from cytidine analog treatment, compared withthe absence of methylation. Influence of methylation level on OS may beassessed in each IPSS cytogenetic subgroup (good, intermediate, andpoor). For example, the influence of methylation on OS may be strongestin the “poor” risk group, where risk of death is greatest.

Such data and analysis may indicate, e.g., that patients with lowerlevels of methylation may derive greater benefit from treatment withpharmaceutical compositions comprising a cytidine analog (e.g.,azacitidine). Molecular biomarkers may be important in MDS, e.g., asindicators of disease prognosis and predictors of response to epigenetictherapy.

I. Example 9

Clinical studies are conducted to assess the ability of an oralformulation comprising a cytidine analog, such as 5-azacytidine, totreat patients having lung cancer, e.g., non-small-cell lung cancer(NSCLC). Such studies may include, e.g., an assessment of the ability tostop or reverse the growth of particular NSCLC cell types in patientshaving NSCLC). In certain clinical studies, patients are tested forparticular NSCLC cell types, e.g., A549, H1975, H522, H23, H460, andH1299, prior to administration of the oral formulation. In certainclinical studies, patients with cell types known or believed to benefitpreferentially from cytidine analog (e.g., 5-azacytidine) administrationmay be enrolled. In certain clinical studies, patients having NSCLC areenrolled without analysis of particular NSCLC cell type. In certainclinical studies, patients having any type of NSCLC cells are candidatesfor treatment with an oral formulation provided herein.

In certain clinical studies, patients from any of the three main NSCLCgroups may be enrolled, i.e., (1) patients with tumors that aresurgically resectable; (2) patients with either locally or regionallyadvanced lung cancer; or (3) patients with distant metastases at thetime of diagnosis. In certain clinical studies, patients may becurrently undergoing additional treatment for NSCLC, including, e.g.,surgery, chemotherapy, or radiation therapy.

In certain clinical studies, patients who are administered an oralformulation comprising a cytidine analog (e.g., 5-azacytidine) may alsobe administered one or more additional therapeutic agents, examples ofwhich are disclosed herein. The additional therapeutic agent(s) may beadministered in the same oral formulation as the cytidine analog, or maybe co-administered (e.g., via PO, SC or IV administration) incombination with an oral formulation comprising the cytidine analog. Theappropriate amount and dosing schedule for an additional therapeuticagent is determined for a particular patient using methods known in theart.

An association between gene methylation and recurrence of NSCLC tumorsis known in the art. See, e.g., M. V. Brock et al., N. Engl. J. Med.,2008, 358(11): 1118-28. Accordingly, in certain clinical studiesprovided herein, patients are screened prior to enrollment and/ormonitored during the trial for DNA or RNA methylation levels, whichindicate a potential response to treatment with an oral formulationcomprising a cytidine analog (e.g., 5-azacytidine). In certain clinicalstudies, patients with high levels of DNA methylation (e.g., CpG islandmethylation) and/or an increased potential for transcriptional silencingof tumor-suppressor genes may be administered a cytidine analog (e.g.,5-azacytidine) known or believed to prevent or reverse hypermethylation(e.g., by reducing the activity of one or more DNA methyltransferaseenzymes). In such studies, patients may also be co-administered one ormore additional therapeutic agents known or believed to reduceepigenetic silencing, such as, e.g., compounds that inihibit histonedeacetylase enzymes (HDACs), which regulate the acetylation anddeacetylation of histone residues that increase or decrease geneexpression. See, e.g., J. G. Herman & S. B. Baylin, N. Engl. J. Med.,2003, 349:2042-54; P. A. Jones & S. B. Baylin, Nature Rev. Gen., 2002,3:415-28. Suitable HDAC inhibitors for co-administration in the clinicalstudies disclosed herein are known in the art and/or described herein(e.g., entinostat or vorinostat).

The amount of cytidine analog (e.g., 5-azacytidine) in the oralformulations administered during the clinical studies depends, e.g., onthe individual characteristics of the patient, including, inter alia,the stage and progression of the patient's NSCLC, the patient's age andweight, the patient's prior treatment regimens, and other variables, asknown in the art. In certain clinical studies, potential starting dosesmay be, e.g., about 60 mg, about 120 mg, about 180 mg, about 240 mg,about 300 mg, about 360 mg, about 420 mg, about 480 mg, about 540 mg,about 600 mg, about 660 mg, about 720 mg, about 780 mg, about 840 mg,about 900 mg, about 960 mg, about 1020 mg, or greater than about 1020 mgof the cytidine analog (e.g., 5-azacytidine) daily for a specified timeperiod, e.g., about 1 week, about 1.5 weeks, about 2 weeks, about 2.5weeks, about 3 weeks, about 3.5 weeks, about 1 month, about 1.5 months,about 2 months, or a longer time period. Other potential starting dosesand time periods are disclosed herein. Cycles may be repeated asdesired, e.g., over a period of one or more months, as disclosed herein.After a certain number of cycles, the dosage may be increased toincrease the beneficial effect, provided such an increase will not causeundesirable toxicity effects. Patients may be treated for a minimumnumber of cycles, as disclosed herein. Complete or partial response mayrequire additional treatment cycles. Treatment may be continued as longas the patient continues to benefit.

J. Example 10

Clinical studies are conducted to assess the ability of an oralformulation comprising a cytidine analog, such as 5-azacytidine, totreat patients having an ovarian cancer (including, e.g., the ability tostop or reverse the growth of cancer cells in patients having an ovariancancer). Particular ovarian cancers include, but are not limited to,ovarian epithelial cancer, ovarian germ cell tumors, and ovarian lowmalignant potential tumors. In certain clinical studies, patients arescreened for the presence of a particular type of ovarian cancer priorto administration of the oral formulation. In certain clinical studies,patients with a type of ovarian cancer known or believed to benefitpreferentially from cytidine analog (e.g., 5-azacytidine) administrationmay be enrolled. In certain clinical studies, patients having ovariancancer are enrolled without screening for particular ovarian cancertypes. In certain clinical studies, patients having any type of ovariancancer are candidates for treatment with an oral formulation providedherein. In certain clinical studies, patients may be currentlyundergoing additional treatment for ovarian cancer, including, e.g.,surgery, chemotherapy, or radiation therapy.

In certain clinical studies, patients who are administered an oralformulation comprising a cytidine analog (e.g., 5-azacytidine) may alsobe administered one or more additional therapeutic agents, examples ofwhich are disclosed herein (e.g., carboplatin). The additionaltherapeutic agent(s) may be administered in the same oral formulation asthe cytidine analog, or may be co-administered (e.g., via PO, SC or IVadministration) in combination with an oral formulation comprising acytidine analog. The appropriate amount and dosing schedule for anadditional therapeutic agent is determined for a particular patientusing methods known in the art.

An association between gene methylation and ovarian cancer is known inthe art. See, e.g., G. Gifford et al., Clin. Cancer Res., 2004,10:4420-26. Accordingly, in certain clinical studies provided herein,patients are screened prior to enrollment and/or monitored during thetrial for DNA or RNA methylation levels, which indicate a potentialresponse to treatment with an oral formulation comprising a cytidineanalog (e.g., 5-azacytidine). In certain clinical studies, patients withhigh levels of DNA methylation (e.g., CpG island methylation) and/or anincreased potential for transcriptional silencing of tumor-suppressorgenes may be administered a cytidine analog (e.g., 5-azacytidine) knownor believed to prevent or reverse hypermethylation (e.g., by reducingthe activity of one or more DNA methyltransferase enzymes). In suchstudies, patients may also be co-administered one or more additionaltherapeutic agents known or believed to reduce epigenetic silencing,such as, e.g., compounds that inihibit histone deacetylase enzymes(HDACs), which regulate the acetylation and deacetylation of histoneresidues that increase or decrease gene expression. See, e.g., J. G.Herman & S. B. Baylin, N. Engl. J. Med., 2003, 349:2042-54; P. A. Jones& S. B. Baylin, Nature Rev. Gen., 2002, 3:415-28. Suitable HDACinhibitors for co-administration in the clinical studies disclosedherein are known in the art and/or described herein (e.g., entinostat orvorinostat).

The amount of cytidine analog (e.g., 5-azacytidine) in the oralformulations administered during the clinical studies depends, e.g., onthe individual characteristics of the patient, including, inter alia,the type, stage, and progression of the patient's ovarian cancer, thepatient's age and weight, the patient's prior treatment regimens, andother variables, as known in the art. n certain clinical studies,potential starting doses may be, e.g., about 60 mg, about 120 mg, about180 mg, about 240 mg, about 300 mg, about 360 mg, about 420 mg, about480 mg, about 540 mg, about 600 mg, about 660 mg, about 720 mg, about780 mg, about 840 mg, about 900 mg, about 960 mg, about 1020 mg, orgreater than about 1020 mg of the cytidine analog (e.g., 5-azacytidine)daily for a specified time period, e.g., about 1 week, about 1.5 weeks,about 2 weeks, about 2.5 weeks, about 3 weeks, about 3.5 weeks, about 1month, about 1.5 months, about 2 months, or a longer time period. Otherpotential starting doses and time periods are disclosed herein. Cyclesmay be repeated as desired, e.g., over a period of one or more months,as disclosed herein. After a certain number of cycles, the dosage may beincreased to increase the beneficial effect, provided such an increasewill not cause undesirable toxicity effects. Patients may be treated fora minimum number of cycles, as disclosed herein. Complete or partialresponse may require additional treatment cycles. Treatment may becontinued as long as the patient continues to benefit.

K. Example 11

Clinical studies are conducted to assess the ability of an oralformulation comprising a cytidine analog, such as 5-azacytidine, totreat patients having a pancreatic cancer (including, e.g., the abilityto stop or reverse the growth of cancer cells in patients havingpancreatic cancer). In certain clinical studies, patients are screenedprior to enrollment for a particular type of pancreatic cancer prior toadministration of the oral formulation. Cellular classifications ofpancreatic cancers are known in the art and include, e.g., duct cellcarcinoma; acinar cell carcinoma; papillary mucinous carcinoma; signetring carcinoma; adenosquamous carcinoma; undifferentiated carcinoma;mucinous carcinoma; giant cell carcinoma; mixed type (ductal-endocrineor acinar-endocrine); small cell carcinoma; cystadenocarcinoma (serousand mucinous types); unclassified; pancreatoblastoma; papillary-cysticneoplasm (Frantz tumor); invasive adenocarcinoma associated with cysticmucinous neoplasm or intraductal papillary mucinous neoplasm; mucinouscystic tumor with dysplasia; intraductal papillary mucinous tumor withdysplasia; and pseudopapillary solid tumor. In certain clinical studies,patients are screened prior to enrollment for a particular stage ofpancreatic cancer (e.g., the size of the tumor in the pancreas, whetherthe cancer has spread, and if so, to what parts of the body) prior toadministration of the oral formulation. In certain clinical studies,pancreatic cancer patients believed to benefit preferentially fromcytidine analog (e.g., 5-azacytidine) administration may be enrolled. Incertain clinical studies, patients having pancreatic cancer are enrolledwithout screening for particular pancreatic cancer types. In certainclinical studies, patients having any type of pancreatic cancer arecandidates for treatment with an oral formulation provided herein. Incertain clinical studies, patients may be currently undergoingadditional treatment for pancreatic cancer, including, e.g., surgery,chemotherapy, or radiation therapy.

In certain clinical studies, patients who are administered an oralformulation comprising a cytidine analog (e.g., 5-azacytidine) may alsobe administered one or more additional therapeutic agents, examples ofwhich are disclosed herein (e.g., gemcitabine). The additionaltherapeutic agent(s) may be administered in the same oral formulation asthe cytidine analog, or may be co-administered (e.g., via PO, SC or IVadministration) in combination with an oral formulation comprising acytidine analog. The appropriate amount and dosing schedule for anadditional therapeutic agent is determined for a particular patientusing methods known in the art.

In certain clinical studies provided herein, patients are screened priorto enrollment and/or monitored during the trial for DNA or RNAmethylation levels, which indicate a potential response to treatmentwith an oral formulation comprising a cytidine analog (e.g.,5-azacytidine). In certain clinical studies, patients with high levelsof DNA methylation (e.g., CpG island methylation) and/or an increasedpotential for transcriptional silencing of tumor-suppressor genes may beadministered a cytidine analog (e.g., 5-azacytidine) known or believedto prevent or reverse hypermethylation (e.g., by reducing the activityof one or more DNA methyltransferase enzymes). In such studies, patientsmay also be co-administered one or more additional therapeutic agentsknown or believed to reduce epigenetic silencing, such as, e.g.,compounds that inihibit histone deacetylase enzymes (HDACs), whichregulate the acetylation and deacetylation of histone residues thatincrease or decrease gene expression. See, e.g., J. G. Herman & S. B.Baylin, N. Engl. J. Med., 2003, 349:2042-54; P. A. Jones & S. B. Baylin,Nature Rev. Gen., 2002, 3:415-28. Suitable HDAC inhibitors forco-administration in the clinical studies disclosed herein are known inthe art and/or described herein (e.g., entinostat or vorinostat).

The amount of cytidine analog (e.g., 5-azacytidine) in the oralformulations administered during the clinical studies depends, e.g., onthe individual characteristics of the patient, including, inter alia,the type, stage, and progression of the patient's pancreatic cancer, thepatient's age and weight, the patient's prior treatment regimens, andother variables, as known in the art. In certain clinical studies,potential starting doses may be, e.g., about 60 mg, about 120 mg, about180 mg, about 240 mg, about 300 mg, about 360 mg, about 420 mg, about480 mg, about 540 mg, about 600 mg, about 660 mg, about 720 mg, about780 mg, about 840 mg, about 900 mg, about 960 mg, about 1020 mg, orgreater than about 1020 mg of the cytidine analog (e.g., 5-azacytidine)daily for a specified time period, e.g., about 1 week, about 1.5 weeks,about 2 weeks, about 2.5 weeks, about 3 weeks, about 3.5 weeks, about 1month, about 1.5 months, about 2 months, or a longer time period. Otherpotential starting doses and time periods are disclosed herein. Cyclesmay be repeated as desired, e.g., over a period of one or more months,as disclosed herein. After a certain number of cycles, the dosage may beincreased to increase the beneficial effect, provided such an increasewill not cause undesirable toxicity effects. Patients may be treated fora minimum number of cycles, as disclosed herein. Complete or partialresponse may require additional treatment cycles. Treatment may becontinued as long as the patient continues to benefit.

L. Example 12

Clinical studies are conducted to assess the ability of an oralformulation comprising a cytidine analog, such as 5-azacytidine, totreat patients having a colorectal cancer (including, e.g., the abilityto stop or reverse the growth of cancer cells in patients having acolorectal cancer). In certain clinical studies, patients are screenedprior to enrollment for a particular type of colorectal cancer prior toadministration of the oral formulation. Histologic types of coloncancers are known in the art and include, e.g., adenocarcinoma; mucinous(colloid) adenocarcinoma; signet ring adenocarcinoma; scirrhous tumors;and neuroendocrine tumors. The World Health Organization classificationof tumors of the colon and rectum include (1) Epithelial Tumors, whichinclude: Adenoma (e.g., tubular, villous, tubulovillous, and serrated);Intraepithelial neoplasia (dysplasia) associated with chronicinflammatory diseases (e.g., low-grade glandular intraepithelialneoplasia and high-grade glandular intraepithelial neoplasia); Carcinoma(e.g., adenocarcinoma, mucinous adenocarcinoma, signet-ring cellcarcinoma, small cell carcinoma, adenosquamous carcinoma, medullarycarcinoma, and undifferentiated carcinoma); Carcinoid(well-differentiated neuroendocrine neoplasm) (e.g., enterochromaffin(EC)-cell, serotonin-producing neoplasm, L-cell, glucagon-like peptideand pancreatic polypeptide/peptide YY (PYY)-producing tumor, andothers); and Mixed carcinoma-adenocarcinoma; and (2) NonepithelialTumors, which include: Lipoma; Leiomyoma; Gastrointestinal stromaltumor; Leiomyosarcoma; Angiosarcoma; Kaposi sarcoma; Melanoma; andothers; as well as Malignant lymphomas (e.g., marginal zone B-celllymphoma of mucosa-associated lymphoid tissue type, mantle celllymphoma, diffuse large B-cell lymphoma, Burkitt lymphoma, andBurkitt-like/atypical Burkitt lymphoma. In certain clinical studies,patients are screened prior to enrollment for a particular stage ofcolorectal cancer (e.g., the size of the tumor in the colon or rectum,whether the cancer has spread, and if so, to what parts of the body)prior to administration of the oral formulation. In certain clinicalstudies, colorectal cancer patients believed to benefit preferentiallyfrom cytidine analog (e.g., 5-azacytidine) administration may beenrolled. In certain clinical studies, patients having a colorectalcancer are enrolled without screening for particular colorectal cancertypes. In certain clinical studies, patients having any type ofcolorectal cancer are candidates for treatment with an oral formulationprovided herein. In certain clinical studies, patients may be currentlyundergoing additional treatment for colorectal cancer, including, e.g.,surgery, chemotherapy, or radiation therapy.

In certain clinical studies, patients who are administered an oralformulation comprising a cytidine analog (e.g., 5-azacytidine) may alsobe administered one or more additional therapeutic agents, examples ofwhich are disclosed herein. The additional therapeutic agent(s) may beadministered in the same oral formulation as the cytidine analog, or maybe co-administered (e.g., via PO, SC or IV administration) incombination with an oral formulation comprising a cytidine analog. Theappropriate amount and dosing schedule for an additional therapeuticagent is determined for a particular patient using methods known in theart.

An association between gene methylation and colorectal cancer is knownin the art. See, e.g., A. M. Jubb et al., J. Pathol., 2001, 195:111-134.Accordingly, in certain clinical studies provided herein, patients arescreened prior to enrollment and/or monitored during the trial for DNAor RNA methylation levels, which indicate a potential response totreatment with an oral formulation comprising a cytidine analog (e.g.,5-azacytidine). In certain clinical studies, patients with high levelsof DNA methylation (e.g., CpG island methylation) and/or an increasedpotential for transcriptional silencing of tumor-suppressor genes may beadministered a cytidine analog (e.g., 5-azacytidine) known or believedto prevent or reverse hypermethylation (e.g., by reducing the activityof one or more DNA methyltransferase enzymes). In such studies, patientsmay also be co-administered one or more additional therapeutic agentsknown or believed to reduce epigenetic silencing, such as, e.g.,compounds that inihibit histone deacetylase enzymes (HDACs), whichregulate the acetylation and deacetylation of histone residues thatincrease or decrease gene expression. See, e.g., J. G. Herman & S. B.Baylin, N. Engl. J. Med., 2003, 349:2042-54; P. A. Jones & S. B. Baylin,Nature Rev. Gen., 2002, 3:415-28. Suitable HDAC inhibitors forco-administration in the clinical studies disclosed herein are known inthe art and/or described herein (e.g., entinostat or vorinostat).

The amount of cytidine analog (e.g., 5-azacytidine) in the oralformulations administered during the clinical studies depends, e.g., onthe individual characteristics of the patient, including, inter alia,the type, stage, and progression of the patient's colorectal cancer, thepatient's age and weight, the patient's prior treatment regimens, andother variables, as known in the art. In certain clinical studies,potential starting doses may be, e.g., about 60 mg, about 120 mg, about180 mg, about 240 mg, about 300 mg, about 360 mg, about 420 mg, about480 mg, about 540 mg, about 600 mg, about 660 mg, about 720 mg, about780 mg, about 840 mg, about 900 mg, about 960 mg, about 1020 mg, orgreater than about 1020 mg of the cytidine analog (e.g., 5-azacytidine)daily for a specified time period, e.g., about 1 week, about 1.5 weeks,about 2 weeks, about 2.5 weeks, about 3 weeks, about 3.5 weeks, about 1month, about 1.5 months, about 2 months, or a longer time period. Otherpotential starting doses and time periods are disclosed herein. After acertain number of cycles, the dosage may be increased to increase thebeneficial effect, provided such an increase will not cause undesirabletoxicity effects. Patients may be treated for a minimum number ofcycles, as disclosed herein. Complete or partial response may requireadditional treatment cycles. Treatment may be continued as long as thepatient continues to benefit.

M. Example 13

Clinical studies are conducted to identify a dose and schedule of anoral formulation of 5-azacytidine that can be safely administered withrituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone(R-CHOP). Clinical studies are conducted to evaluate the safety andmaximum tolerated dose (MTD) or the maximal administered dose (MAD) ofthe oral formulation of 5-azacytidine in combination with rituximab,cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) insubjects with high risk (IPI 3 or more) previously untreated diffuselarge B-cell lymphoma (DLBCL) or Grade 3B follicular lymphoma (FL).Clinical studies are also conducted to determine pharmacokinetics (PK)of the oral formulation of 5-azacytidine when administered alone and incombination with R-CHOP and to explore preliminary efficacy of the oralformulation of 5-azacytidine plus R-CHOP by 2007 International WorkingGroup (IWG) criteria. Clinical studies are also conducted to evaluatethe pharmacodynamic (PD) effects of oral formulation of 5-azacytidine,to evaluate potential predictive/correlative biomarkers for DLBCLsubgroups that are considered important and to explore cytidinedeaminase (CDA) activity and polymorphisms.

A further purpose of this study is to evaluate the safety of oralformulation of 5-azacytidine when combined with R-CHOP. The otherpurposes of this study are to understand how the human body takes in andremoves oral formulation of 5-azacytidine when given with R-CHOP, if theDiffuse large B-cell lymphoma or Grade 3B follicular lymphoma tumor sizedecreases, and to look into the effects the drug can have on the humanbody.

The treatment period starts when the subject receives the study drug.The maximum time the subject receive study treatment is 5 months. Theintent is for the subject to complete 6 cycles of treatment. Each cyclewill be 21 days. Rituximab, cyclophosamide, doxorubicin, and vincristineare administered on Day 1; while prednisone is administered Days 1-5.

The follow-up period starts when the treatment is completed, changed ordiscontinued for any reason. The subject will have fewer exams, testsand visits. These visits will be every 6 months for up to 2 years.

N. Example 14

Clinical studies in subjects with mantel cell lymphoma (MCL) areconducted to identify a dose and schedule of an oral formulation of5-azacytidine that can be safely administered with rituximab,cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP).Clinical studies are conducted to evaluate the safety and maximumtolerated dose (MTD) or the maximal administered dose (MAD) of the oralformulation of 5-azacytidine in combination with rituximab,cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) insubjects with mantel cell lymphoma (MCL). Clinical studies are alsoconducted to determine pharmacokinetics (PK) of the oral formulation of5-azacytidine when administered alone and in combination with R-CHOP andto explore preliminary efficacy of the oral formulation of 5-azacytidineplus R-CHOP by 2007 International Working Group (IWG) criteria. Clinicalstudies are also conducted to evaluate the pharmacodynamic (PD) effectsof oral formulation of 5-azacytidine, to evaluate potentialpredictive/correlative biomarkers for mantel cell lymphoma subgroupsthat are considered important and to explore cytidine deaminase (CDA)activity and polymorphisms.

A further purpose of this study is to evaluate the safety of oralformulation of 5-azacytidine when combined with R-CHOP. The otherpurposes of this study are to understand how the human body takes in andremoves oral formulation of 5-azacytidine when given with R-CHOP, if themantel cell lymphoma tumor size decreases, and to look into the effectsthe drug can have on the human body.

The treatment period starts when the subject receives the study drug.The maximum time the subject receive study treatment is 5 months. Theintent is for the subject to complete 6 cycles of treatment. Each cyclewill be 21 days. Rituximab, cyclophosamide, doxorubicin, and vincristineare administered on Day 1; while prednisone is administered Days 1-5.

The follow-up period starts when the treatment is completed, changed ordiscontinued for any reason. The subject will have fewer exams, testsand visits. These visits will be every 6 months for up to 2 years.

The present disclosure has been described in connection with certainembodiments and examples; however, unless otherwise indicated, theclaimed invention should not be unduly limited to such specificembodiments and examples.

1. (canceled)
 2. A compound of the formula:

or a pharmaceutically acceptable salt or solvate thereof, wherein: Y⁴ isa hydrogen isotopically enriched with deuterium, and Y¹, Y², Y³, Y⁵, Y⁶,and Y⁷ are non-enriched hydrogen atoms.